Molecular cloning and functional characterization of an isoflavone glucosyltransferase from Pueraria thomsonii / 中国天然药物

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.

Puerarin Inhibits the Proliferation,Invasion,and Migration of Non-small Cell Lung Cancer Cells through Regulating miR-490/Denticleless E3 Ubiquitin Protein Ligase / 中国医学科学院学报

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.

Mechanism of puerarin reversing SH-SY5Y cell injury induced by Aβ_(1-42) based on proteomics / 中国中药杂志

Puerarin has the anti-Alzheimer's disease (AD) activity,which can reverse nerve injury induced by Aβand inhibit neuronal apoptosis.However,its potential pharmacodynamic mechanism still needs to be further researched.The occurrence and development of AD is due to the change of multiple metabolic links in the body,which leads to the destruction of balance.Puerarin may act on multiple targets and multiple metabolic processes to achieve therapeutic purposes.Quantitative proteomic analysis provides a new choice to understand the mechanism as completely as possible.This research adopted SH-SY5Y cells induced by Aβ_(1-42)to establish AD cell model,and Aβimmunofluorescence detection showed that Aβdecreased significantly after puerarin intervention.The mechanism of puerarin reversing SH-SY5Y cell injured by Aβ_(1-42)was further explored by using label-free non-labeled quantitative technology and Western blot detection based on bioinformatics analysis result.The results showed that most of the differential proteins were related to biological processes such as cellular component organization or biogenesis,cellular component organization and cellular component biogenesis,and they mainly participated in the top ten pathways of P value such as pathogenic Escherichia coli infection,m TOR signaling pathway,regulation of autophagy,regulation of actin cytoskeleton,spliceosome,hepatocellular carcinoma,tight junction,non-small cell lung cancer,apoptosis and gap junction.Annexin V/PI flow cytometry and TUNEL were used to detect apoptosis,and the results showed that Aβdecreased significantly and the rate of apoptosis decreased significantly after puerarin intervention.Western blot analysis found that the protein expression level of autophagy related protein LC3Ⅱwas up-regulated after Aβinduction,and the degree of this up-regulation was further enhanced in puerarin intervention group.The trend of the ratio of LC3Ⅱ/LC3Ⅰamong groups was the same as the protein expression level of LC3Ⅱ，the protein expression level of p62 in the control group,AD model group and puerarin intervention group decreased successively.Protein interaction network analysis showed that CAP1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.Western blot showed that the expressions of p-ERK1/2,Bax and CAP1 were significantly down-regulated and the protein expression level of Bcl-2 was significantly up-regulated after puerarin intervention.Therefore,puerarin might improve the SH-SY5Y cells injured by Aβ_(1-42)through the interaction of multiple biological processes and pathways in cells multiple locations,and CAP1 might play an important role among them.

Research advances in chemical constituents and pharmacological activities of Pueraria genus / 中国中药杂志

Kudzu plants in the subfamily sphenoideae of Leguminosae are commonly used herbs in China, Japan, Korea, India and Thailand, with a long history of medicinal use. They are recorded in Chinese Pharmacopoeia, Japanese Pharmacopeia, Korea Pharmacopeia, Ayurveda Pharmacopoeia of India and Flora of Thailand. There are 15-20 species of Pueraria in the world, including 7 species and 2 varieties in China. At present, there are 6 species with medicinal value, such as Pueraria lobata and P. thomsonii. The main chemical components of the genus are isoflavones, flavonoids, terpenes, steroids, coumarins, puerarin glycosides and benzopyrans. A total of 240 compounds have been isolated and identified from this genus, and their pharmacological effects mainly include improvement of the cardiovascular system, antioxidant, hypoglycemic, antipyretic, anti-inflammatory, anti-alcoholic and estrogen-like effects. In this study, chemical constituents and pharmacological activities of Pueraria at home and abroad were systematically summarized, in order to provide references for the material basis, quality control and further development of Pueraria genus.

The influence of phytoestrogens or estrogens on the proliferation of the rat endocervical mucosa

SUMMARY INTRODUCTION Although estrogen therapy is widely used against post-menopausal symptoms, it can present adverse effects, including endometrial cancer. Soy isoflavones are considered a possible alternative to estrogen therapy. However, there are still concerns whether isoflavones exert trophic effects on the uterine cervix. OBJECTIVES To evaluate the histomorphometric and immunohistochemical alterations in the uterine cervix of ovariectomized rats treated with soy isoflavones (Iso). METHODS Fifteen adult Wistar rats were ovariectomized (Ovx) and divided into three groups: Group I (Ovx), administered with vehicle solution; Group II (OVX-Iso), administered with concentrated extract of Iso (150 mg/kg) by gavage; and Group III (OVX-E2), treated with 17β-estradiol (10 µg/kg), subcutaneously. After 30 days of treatments, the uterine cervix was fixed in 10% formaldehyde and processed for paraffin-embedding. Sections were stained with Hematoxylin and eosin for morphological and morphometric studies or subjected to immunohistochemistry for detections of Ki-67 and vascular endothelial growth factor-A (Vegf-A). The data obtained were subjected to statistical analysis (p ≤ 0.05). RESULTS We noted an atrophic uterine cervix in GI, whereas it was more voluminous in GII and even more voluminous in GIII. The thickness of the cervical mucosa was significantly higher in GIII, as compared to GI and GII. The cell proliferation (Ki-67) was significantly elevated in the estradiol and isoflavones treated groups, whereas Vegf-A immunoexpression was significantly higher in GIII, as compared to groups GII and GI. CONCLUSIONS Soy isoflavones cause less trophic and proliferative effects in the uterine cervix of rats as compared to estrogen.

RESUMO INTRODUÇÃO Embora a terapia estrogênica seja amplamente utilizada contra sintomas pós-menopausais, ela pode apresentar efeitos adversos, incluindo câncer de mama e endometrial. Assim, as isoflavonas da soja são consideradas uma alternativa possível à terapia estrogênica. No entanto, ainda há controvérsias se estes compostos exercem efeitos tróficos significativos no colo do útero. OBJETIVOS Avaliar as alterações histomorfométricas e imuno-histoquímicas no colo do útero de ratas ovariectomizadas tratadas com isoflavonas da soja (iso). MÉTODOS Quinze ratas Wistar adultas foram ovariectomizadas bilateralmente (Ovx) e separadas em três grupos: Grupo I (Ovx) - veículo (propilenoglicol); Grupo II (Ovx-Iso) - receberam extrato concentrado de Iso (150 mg/kg) e Grupo III (Ovx-E2) - tratado com 17β-estradiol (10 µg/kg); as soluções foram administradas via gavagem por 30 dias consecutivos. Posteriormente, os colos uterinos foram retirados, fixados em formaldeído a 10% tamponado e processados para inclusão em parafina. Cortes (4 µm) foram coradas com hematoxilina e eosina para estudo morfológico e morfométricos, enquanto outros foram submetidos à imuno-histoquímica para detecção de Ki-67 e do fator de crescimento endotelial vascular-A (Vegf-A). Os dados obtidos foram submetidos à análise estatística (p≤0,05). RESULTADOS Observamos a presença de colo uterino atrófico no GI (Ovx), sendo este mais volumoso no GII (Ovx+Iso) e ainda mais volumoso no GIII (Ovx+E2). A espessura da mucosa cervical foi significativamente maior no GIII (Ovx-E2), em comparação ao GI (Ovx) e ao GII (Ovx-Iso). A proliferação celular (Ki-67) foi significativamente mais elevada nos grupos tratados com estradiol e isoflavonas, enquanto a imunoexpressão de Vegf-A foi significativamente maior no GIII (Ovx-E2), em comparação ao GII (Ovx-Iso) e ao GI (Ovx-E2). CONCLUSÕES As isoflavonas da soja causam menos efeitos tróficos e proliferativos no colo do útero de ratas em comparação ao estrogênio.

The cardiovascular protective effect and mechanism of calycosin and its derivatives / 中国天然药物

Cardiovascular disease is the main cause of mortality and morbidity in the world, especially in developing countries. Drug therapy is one of the main ways to treat cardiovascular diseases. Among them, great progress has been made in the treatment of cardiovascular diseases with traditional Chinese medicine. In terms of experimental research, the mechanism of traditional Chinese medicine in the treatment of cardiovascular diseases has been thoroughly discussed in vitro and in vivo. In terms of clinical treatment, traditional Chinese medicine with flavonoids, saponins and alkaloids as the main effective components has a definite effect on the treatment of cardiovascular diseases such as arrhythmia, myocardial ischemia, angina pectoris and myocardial infarction, with high safety and good application prospects. With the further research on the effective ingredients, mechanism and adverse reactions of traditional Chinese medicine, it will be beneficial to the effectiveness of traditional Chinese medicine, reduce side effects and promote the modernization of traditional Chinese medicine. Calycosin and its derivatives, the main bioactive flavonoids in Astragalus membranaceus have multiple biological effects, such as antioxidant, pro-angiogenesis, anti-tumour, and anti-inflammatory effects. Based on the above biological effects, calycosin has been shown to have good potential for cardiovascular protection. The potent antioxidant effect of calycosin may play an important role in the cardiovascular protective potential. For injured cardiac myocytes, calycosin and its derivatives can alleviate the cell damage mainly marked by the release of myocardial enzymes and reduce the death level of cardiac myocytes mainly characterized by apoptosis through various mechanisms. For vascular endothelial cells, calycosin also has multiple effects and multiple mechanisms, such as promoting vascular endothelial cell proliferation, exerting vasodilating effect and directly affecting the synthesis function of endothelial cells. The present review will address the bioactivity of calycosin in cardiovascular diseases such as protective effects on cardiac myocytes and vascular endothelial cells and elucidate main mechanism of calycosin and its derivatives to exert the above biological effects.

Glabridin alleviates ischemia/reperfusion-induced functional failure of smooth muscle of rat ileum by upregulating the cAMP / Glabridin alivia la falla funcional inducida por isquemia/reperfusión del músculo liso de ileum murino regulando el incremento de cAMP

Despite the development of modern medicine, alternative medicine, which has not lost its timeliness, remains attractive for the treatment of various diseases. Glabridin, a major flavonoid of Glycyrrhiza glabra, is known for its antioxidant and anti-inflammatory activity. The aim of this study was: 1) to determine the possible protective role of glabridin against ischemia/reperfusion (I/R) injury of the intestine; 2) to evaluate the in vitrocontractile responses of ileum smooth muscles to acetylcholine after an intestinal I/R; and 3) to explain the underlying molecular mechanism of its effect. Rats were assigned to groups of six rats each; 1) I/R, 2) gla10, 3) gla20, 4) gla40, 5) N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester monohydrochloride (L-NAME)+gla40, and 6) Sham group. The healing effect of glabridin was abolished by L-NAME. Glabridin did not cause contractility of the smooth muscles to acetylcholine-induced contractile responses in intestinal I/R. Yet, it increased to spontaneous basal activity.

A pesar del desarrollo de la medicina moderna, la medicina alternativa, sin perder su vigencia, sigue siendo atractiva para el tratamiento de varias enfermedades. Glabradina, el flavonoide mayoritario de Glycyrrhiza glabra, es conocido por su actividad antioxidante y antiinflamatoria. Los propósitos de este estudio fueron: 1) Determinar el posible rol protector de glabradina ante daños intestinales por isquemia/reperfusion (I/R) 2) Evaluar in vitrolas respuestas de contracción de los músculos lisos del ileum ante acetilcolina después de I/R intestinal; y 3) Explicar el mecanismo molecular subyacente de este efecto. Se asignaron grupos de seis ratas: 1) I/R, 2) gla10, 3) gla20, 4) gla40, 5) N5-[imino(nitroamino)metil]-L-ornithina, metil ester monohidrochloruro (L-NAME)+gla40, y 6) Grupo testigo. El efecto curativo de glabridina fue abolido por L-NAME. Glabridina no causó contracción en el músculo liso como respuesta acetilcolina-inducida I/R. Además, incrementa la actividad basal expontánea.

Puerarin inhibits hepatocellular carcinoma invasion and metastasis through miR-21-mediated PTEN/AKT signaling to suppress the epithelial-mesenchymal transition

Hepatocellular carcinoma (HCC) is one of the most common primary malignant tumors of the liver worldwide. Liver resection and transplantation are currently the only effective treatments; however, recurrence and metastasis rates are still high. Previous studies have shown that the epithelial-mesenchymal transition (EMT) is a key step in HCC invasion and metastasis. Inhibition of EMT has become a new therapeutic strategy for tumors. Recently, puerarin, a well-characterized component of traditional Chinese medicine, has been isolated from Pueraria radix and exerts positive effects on many diseases, particularly cancers. In this study, CCK-8, EdU immunofluorescence, colony formation, wound healing, and migration assays were used to detect the effects of puerarin on HCC cells. We further analyzed the relationship between puerarin and miR-21/PTEN/EMT markers in HCC cell lines. Our results showed that HCC cell proliferation, migration, invasion, tumor formation, and metastasis were reduced by puerarin in vitro and in vivo. Additionally, puerarin inhibited the EMT process of HCC by affecting the expression of Slug and Snail. Moreover, oncogenic miR-21 was inhibited by puerarin, coupled with an increase in the tumor suppressor gene PTEN. Increasing miR-21 expression or decreasing PTEN expression reversed the inhibition effects of puerarin in HCC. These data confirmed that puerarin affects HCC through the miR-21/PTEN/EMT regulatory axis. Overall, puerarin may represent a chemopreventive and/or chemotherapeutic agent for HCC treatment.

Methylophiopogonanone A, an Ophiopogon homoisoflavonoid, alleviates high-fat diet-induced hyperlipidemia: assessment of its potential mechanism

Methylophiopogonanone A (MO-A), a homoisoflavonoid extracted from Ophiopogon japonicus, has been shown to attenuate myocardial apoptosis and improve cerebral ischemia/reperfusion injury. However, the hypolipidemic effects remain unknown. This study was performed to investigate a potential hypolipidemic effect of MO-A in hyperlipidemia rats, as well as its underlying mechanism of action. A rat model of hyperlipidemia was induced by a high-fat diet (HFD). Animals were randomly divided into three groups (n=8/group): normal control group (NC), HFD group, and HFD+MO-A (10 mg·kg-1·d-1) treatment group. The effects of MO-A on serum lipids, body weight, activity of lipoprotein metabolism enzyme, and gene expression of lipid metabolism were evaluated in HFD-induced rats. In HFD-induced rats, pretreatment with MO-A decreased the body weight gain and reduced serum and hepatic lipid levels. In addition, pretreatment with MO-A improved the activities of lipoprotein lipase and hepatic lipase in serum and liver, down-regulated mRNA expression of acetyl CoA carboxylase and sterol regulatory element-binding protein 1c, and up-regulated mRNA expression of low-density lipoprotein receptor and peroxisome proliferator-activated receptor α in the liver. Our results indicated that MO-A showed strong ability to ameliorate the hyperlipidemia in HFD-induced rats. MO-A might be a potential candidate for prevention of overweight and dyslipidemia induced by HFD.

Differential formononetin content in cultivars and experimental lines of red clover (Trifolium pratense L. ) plants affect the feeding behaviour of Hylastinus obscurus (Coleoptera: Curculionidae) / Contenido diferencial de formononetina en cultivares y líneas experimentales de plantas de trébol rosado (Trifolium pratense L. ) afecta la conducta alimenticia de Hylastinus obscurus (Coleoptera: Curculionidae)

Red clover (Trifolium pratense L.) is a perennial plant widely used as a forage resource for several animals. This plant is the exclusive host of Hylastinus obscurus (Marsham) which causes irreparable damages to the root system affecting their persistence. It has been reported that the presence of the isoflavonoid formononetin in roots of red clover could act as an antifeedant on H. obscurus. There are not studies related to the formononetin content in red clover roots to the antifeedant effect elicited by experimental lines and cultivar of red clover. Six red clover genotypes were investigated in both formononetin content and their respective antifeedant action. The results showed to Sabtoron High and Superqueli-INIA with both the highest formononetin content in red clover roots and antifeedant effect, allowing to suggest that this secondary metabolites could be used as a chemical factor for red clover plants. Moreover, a rapid methodology for searching red clover genotypes with high formononetin content is reported.

El trébol rosado (Trifolium pratense L.) es una planta perenne ampliamente utilizada como fuente de forraje de variados animales. Esta planta es el exclusivo hospedero de Hylastinus obscurus (Marsham) el cual causa irreparables daños al sistema radical afectando seriamente su persistencia. Se ha reportado que la presencia del isoflavonoide formononetina en raíces del trébol rosado podría actuar como antialimentario sobre H. obscurus. Actualmente no existen estudios que relacionen el contenido de formononetina en raíces de trébol rosado con el efecto antialimentario elicitado por líneas experimentales y cultivares de trébol rosado. Seis genotipos de esta leguminosa fueron evaluados en cuanto a su contenido de formononetina y actividad antialimentaria. Los resultados mostraron que los cultivares Sabtoron High y Superqueli-INIA presentaron altos niveles de formononetina en sus raíces y efecto antialimentario sobre H. obscurus, lo que permite sugerir que este metabolito secundario podría ser usado como factor químico para incrementar la persistencia de plantas de trébol rosado. Además, se informa una metodología rápida para la búsqueda de genotipos con altos contenidos de formononetina.

Antifungal activity against colletotrichum acutatum and colletotrichum gloeosporioides of the major constituents from wood sawdust of platymiscium gracile benth / Actividad antifúngica contra colletotrichum acutatum y colletotrichum gloeosporioides de los constituyentes mayoritarios del aserrín de madera de platymiscium gracile benth

The tree tomato (Solanum betaceum Cav., Solanaceae) anthracnose, caused by the fungi Colletotrichum acutatum and Colletotrichum gloeosporioides, is the most important disease of this crop in Colombia for its wide distribution and the losses it causes. In the present work, the in vitro antifungal activity of the soluble fractions in n-hexane, dichloromethane, and ethyl acetate, and their major constituents from the sawdust of timber specie Platymiscium gracile Benth. (Fabaceae) against both fungi was evaluated. The n-hexane-soluble fraction exhibited the greatest inhibitory effect. The metabolites homopterocarpin (a pterocarpan, 0.39 percent dry weight), calycosin (an isoflavone, 2.01 percent) and scoparone (a coumarin, 1.48 percent) were isolated for the first time from wood sawdust of P. gracile. The structure of these compounds was determined by 1H and 13C NMR analyses. The three compounds tested showed significant antifungal activity.

La antracnosis del tomate de árbol (Solanum betaceum Cav., Solanaceae), ocasionada por los hongos Colletotrichum acutatum y Colletotrichum gloeosporioides, es la enfermedad más importante de este cultivo en Colombia por su amplia distribución y las pérdidas que ocasiona. En el presente trabajo se evaluó la actividad antifúngica in vitro de las fracciones solubles en n-hexano, diclorometano y acetato de etilo, y sus componentes mayoritarios, del aserrín de la especie maderable Platymiscium gracile Benth. (Fabaceae), contra ambos hongos. La fracción en n-hexano exhibió el mayor efecto inhibitorio. Los metabolitos homopterocarpina (un pterocarpano; 0.39 por ciento del peso seco de aserrín), calicosin (una isoflavona; 2.01 por ciento) y escoparona (una cumarina; 1.48 por ciento) se aislaron por primera vez desde el aserrín de madera de P. gracile empleando técnicas cromatográficas. La estructura de los compuestos se determinó por análisis de RMN de 1H y 13C. Los tres metabolitos mostraron una actividad antifúngica significativa contra ambos hongos.

Isoflavone formononetin from red propolis acts as a fungicide against Candida sp

Abstract A bioassay-guided fractionation of two samples of Brazilian red propolis (from Igarassu, PE, Brazil, hereinafter propolis 1 and 2) was conducted in order to determine the components responsible for its antimicrobial activity, especially against Candida spp. Samples of both the crude powdered resin and the crude ethanolic extract of propolis from both locations inhibited the growth of all 12 tested Candida strains, with a minimum inhibitory concentration of 256 µg/mL. The hexane, acetate and methanol fractions of propolis 1 also inhibited all strains with minimum inhibitory concentration values ranging from 128 to 512 µg/mL for the six bacteria tested and from 32 to 1024 µg/mL for the yeasts. Similarly, hexane and acetate fractions of propolis sample 2 inhibited all microorganisms tested, with minimum inhibitory concentration values of 512 µg/mL for bacteria and 32 µg/mL for yeasts. The extracts were analyzed by HPLC and their phenolic profile allowed us to identify and quantitate one phenolic acid and seven flavonoids in the crude ethanolic extract. Formononetin and pinocembrin were the major constituents amongst the identified compounds. Formononetin was detected in all extracts and fractions tested, except for the methanolic fraction of sample 2. The isolated isoflavone formononetin inhibited the growth of all the microorganisms tested, with a minimum inhibitory concentration of 200 µg/mL for the six bacteria strains tested and 25 µg/mL for the six yeasts. Formononetin also exhibited fungicidal activity against five of the six yeasts tested. Taken together our results demonstrate that the isoflavone formononetin is implicated in the reported antimicrobial activity of red propolis.

A vigilância e o monitoramento das principais doenças crônicas não transmissíveis no Brasil - Pesquisa Nacional de Saúde, 2013 / Surveillance and monitoring of major chronic diseases in Brazil - National Health Survey, 2013

RESUMO: Objetivo: Descrever as principais doenças crônicas não transmissíveis (DCNT) no país segundo as informações coletadas em indivíduos de 18 anos ou mais de idade. Métodos: Foram utilizados dados da Pesquisa Nacional de Saúde (PNS), 2013, estudo transversal de base populacional. As proporções de cada DCNT foram calculadas e apresentadas segundo sexo, com intervalo de confiança de 95% (IC95%), com os valores absolutos. Resultados: Do total de entrevistados, 45,1% referiram ter pelo menos uma DCNT. A Região com maior prevalência de DCNT foi a Sul (52,1%). A hipertensão arterial apresentou a maior prevalência dentre as DCNT, com 21,4%, seguida por problema crônico de coluna (18,5%), depressão (7,6%), artrite (6,4%) e diabetes (6,2%). O grau de limitação intenso/muito intenso apresentou maiores prevalências para outra doença mental (37,6%) e acidente vascular cerebral (AVC) (25,5%). Conclusão: A melhoria dos serviços de saúde é indispensável para uma resposta efetiva à dupla carga de adoecimento de países de média e baixa renda.

ABSTRACT: Objective: To describe the major noncommunicable diseases (NCDs) in Brazil, according to the information collected from individuals aged 18 years or older. Methods: Data from the National Health Survey (PNS), 2013, a transversal population-based study, were used. The proportions of each NCD were calculated and presented according to sex, with a 95% confidence interval (95%CI), with the absolute values. Results: Of the total respondents, 45.1% reported presenting at least one NCD. The region with the highest prevalence of NCDs was the South (52.1%). Hypertension showed the highest prevalence among NCDs, with 21.4%, followed by chronic back problem (18.5%), depression (7.6%), arthritis (6.4%), and diabetes (6.2%). The intense/very intense degree of limitation showed a higher prevalence of other mental illnesses (37.6%) and cerebrovascular accident (25.5%). Conclusion: The improvement of health services is essential for an effective response to the double burden of illness in the middle- and low-income countries.

Isolation and characterization of homoisoflavonoids from Scilla persica HAUSSKN

abstract Medicinal plants have many traditional claims including the treatment of ailments of infectious origin. In the evaluation of traditional claims, scientific research is extremely important. In this study, five homoisoflavonoids named 3-(4'-hydroxybenzylidene)-5,7-dihydroxy-6-methoxychroman-4-one(Autumnalin),3-(4'-hydroxybenzyl)-5,7-dihydroxy-6-methoxychroman-4-one (3,9-dihydro-autumnalin), 3-(3',4'-dihydroxybenzyl)-5,8-dihydroxy-7-methoxychroman-4-one,3-(3',4'-dihydroxybenzylidene)-5,8-dihydroxy-7-methoxychroman-4-one and 3-(3',4'-dihydroxybenzylidene)-5,7-dihydroxy-6-methoxychroman-4-one, were isolated from the bulbs of the plant Scilla persica HAUSSKN. Their structures were established on the basis of extensive spectroscopic analyses such as NMR, MS, IR and UV.

resumo Plantas medicinais apresentam muitas atribuições tradicionais, incluindo o tratamento de doenças de origem infecciosa. A pesquisa científica é extremamente importante na avaliação dos usos tradicionais. Neste estudo, cinco homoisoflavonóides: 3-(4'-hidroxibenzilideno)-5,7-diidroxi-6-metoxicroman-4-ona(autumnalina), 3-(4'-hidroxibenzil)-5,7-diidroxi-6- metoxicroman-4-ona (3,9-diidro-autumnalina), 3-(3',4'-diidroxibenzil)-5,8-diidroxi-7- metoxicroman-4-ona, 3-(3',4'-diidroxibenzilideno)-5,8-diidroxi-7- metoxicroman-4-ona e 3-(3',4'-diidroxibenzilideno)-5,7-diidroxi-6- metoxicroman-4-ona foram isolados dos bulbos da planta Scilla persica HAUSSKN. Suas estruturas foram estabelecidas com base na extensa análise espectroscópica, como RMN, EM, IV e UV.

Puerarin protects against damage to spatial learning and memory ability in mice with chronic alcohol poisoning

We evaluated the effect of puerarin on spatial learning and memory ability of mice with chronic alcohol poisoning. A total of 30 male C57BL/6 mice were randomly divided into model, puerarin, and control groups (n=10 each). The model group received 60% (v/v) ethanol by intragastric administration followed by intraperitoneal injection of normal saline 30 min later. The puerarin group received intragastric 60% ethanol followed by intraperitoneal puerarin 30 min later, and the control group received intragastric saline followed by intraperitoneal saline. Six weeks after treatment, the Morris water maze and Tru Scan behavioral tests and immunofluorescence staining of cerebral cortex and hippocampal neurons (by Neu-N) and microglia (by Ib1) were conducted. Glutamic acid (Glu) and gamma amino butyric acid (GABA) in the cortex and hippocampus were assayed by high-performance liquid chromatography (HPLC), and tumor necrosis factor (TNF)-α and interleukin (IL)-1β were determined by ELISA. Compared with mice in the control group, escape latency and distance were prolonged, and spontaneous movement distance was shortened (P<0.05) by puerarin. The number of microglia was increased in both the cortex and hippocampal dentate gyrus (P<0.01), and neurons were reduced only in the hippocampal dentate gyrus (P<0.01) in puerarin-treated mice. In the model group, Glu and GABA levels decreased (P<0.05), and Glu/GABA, TNF-α, and IL-1β increased (P<0.01) with puerarin treatment, returning to near normal levels. In conclusion, puerarin protected against the effects of chronic alcohol poisoning on spatial learning and memory ability primarily because of anti-inflammatory activity and regulation of the balance of Glu and GABA.

Equol and daidzein decrease migration, invasion and matrix metalloproteinase (MMPs) gene expression in prostate cancer cell lines, DU-145 and PC-3 / Equol y daidzeina disminuyen la migración, invasión y la expresión génica de metaloproteinasa de la matriz(MMPs) en las líneas celulares prostáticas cancerosas DU-145 y PC-3

This search is focused on the study of diet compounds that may have any potential chemopreventive effect against cancer. Some compounds that fulfill this requirement are phytoestrogens. Among them we find genistein (1), the most studied, daidzein (2) and equol (3) (figure 1). To compare the sensitivities of different prostate cancer cells to phytoestrogen treatment, sulphorhodamine B dye assay was performed to determine cell viability. DU-145 and PC-3 prostate cancer cell lines treated with various doses of phytoestrogen (0-12.5-25-50 and 100 μM) for different times (24, 48 and 72h). For cell invasion or migration assay cells were seeded in a Transwell chamber with or without coating Matrigel respectively. DU-145 and PC-3 cells were treated previously with phytoestrogen (50 μM) for 24h. The study showed that equol, daidzein and genistein inhibited migration and invasion in prostate cancer cell lines. Moreover, we analyzed the effects of phytoestrogens in MMP-2 and MMP-9 mRNA expression by RT-PCR. The results indicated that equol, daidzein and genistein diminished the expression of MMP-2 and MMP-9 in a cell-dependent manner. Our data suggested that equol, daidzein and genistein inhibited migration and invasion in prostate cancer cell lines. Moreover, the results also suggest that down-regulation of MMP-2 and MMP- 9 might be involved in the inhibition of invasion of PC-3 and DU-145 cells after genistein, daidzein and equol treatment.

Este trabajo se centra en el estudio de los compuestos de dieta que pueden tener potencial efecto quimiopreventivo contra el cáncer. Algunos de estos compuestos son los fitoestrógenos. Entre ellos encontramos la genisteína (1), el más estudiado, la daidzeína (2) y el equol (3) (figura 1). Para comparar el efecto de estos fitoestrogenos sobre las líneas celulares de cáncer de próstata, DU-145 y PC-3, se utilizó el ensayo de sulforodamina B para determinar la viabilidad celular tras los tratamientos con diferentes concentraciones de fitoestrógenos (0-12.5-25-50-100 μM) durante diferentes tiempos (24, 48, 72 h). Para analizar el efecto sobre la migración celular, las células DU-145 y PC-3 fueron tratadas previamente con una concentración de fitoestrógrno (50 μM) durante 24 horas y sembradas en una cámara Transwell sin recubrir. El estudio mostró que el equol, daidzeína y genisteína inhibió en MMP-2 y MMP-9 expresiones de genes en líneas celulares de cáncer de próstata, la PC-3 y DU-145. Los resultados indicaron que la daidzeína disminuyó la expresión de MMP- 2 y MMP-9 en DU-145 células. Nuestros datos sugieren que equol, daidzeína y genisteína inhiben la migración y la invasión de líneas celulares de cáncer de próstata.

Morfologia dos cardiomiócitos e quantificação do colágeno no miocárdio de ratas tratadas com isoflavonas ou estrogênios / Cardiomyocytes morphology and collagen quantification in the myocardium of female rats treated with isoflavones or estrogens

OBJETIVOS: Avaliar a morfologia dos cardiomiócitos e quantificar o colágeno presente no miocárdio de ratas tratadas com extrato concentrado de soja ou 17β-estradiol (E2). MÉTODOS: Vinte e oito ratas foram divididas em quatro grupos: GCtrl - fase de estro; GOvx - ovariectomizadas (Ovx); GIso - Ovx tratadas com extrato de soja (150 mg/kg, por dia); GE2 - Ovx tratadas com E2 (10 µg/kg, por dia). As drogas e o veículo (0,2 mL de propilenoglicol) foram administrados após 30 dias da realização da ovariectomia, por 30 dias consecutivos. No último dia os animais foram anestesiados, o coração retirado, mergulhado em formaldeído a 10%, e fragmentos dos ventrículos submetidos a processamento histológico, sendo os cortes corados pela hematoxilina e eosina ou pelo picrosirius-red. As análises histomorfométricas (contagem, volume nuclear e quantificação do colágeno) foram realizadas em microscópio de luz e software AxioVision Rel. 4.2, sendo o colágeno determinado pelo programa Imagelab 2000. Os dados foram submetidos ao teste de ANOVA complementado pelo teste de Tukey (p<0,05). RESULTADOS: Notamos maior quantidade de núcleos de cardiomiócitos nos animais dos grupos Ovx e Iso do que no GE2 e GCtrl (GOvx=121,7±20,2=GIso=92,8±15,4>GE2=70,5±14,8=GCtrl=66,3±9,6; p<0,05), sendo o volume nuclear maior nos animais do grupo Ctrl e E2 (GE2=35,7±4,8=GCtrl=29,9±3,6>GIso=26,5±4,5=GOvx=22,4±2,9; p<0,05). Com relação ao colágeno notamos maior concentração no grupo Ovx (GOvx=5,4±0,1>GCtrl=4,0±0,1=GIso=4,4±0,08=GE2=4,3±0,5; p<0,05). CONCLUSÕES: Os estrogênios previnem a diminuição do volume nuclear dos cardiomiócitos e a deposição de colágeno entre as fibras musculares cardíacas. Já a administração de isoflavonas previne somente a deposição de colágeno, o que pode preservar as propriedades mecânicas das fibras cardíacas.

PURPOSES: To evaluate the histomorphometry of cardiomyocytes and collagen present in the myocardium of rats treated with a concentrated extract of soy or 17β-estradiol (E2). METHODS: Twenty-eight rats were divided into four groups: GCtrl - estrus phase; GOvx - ovariectomized (Ovx) and receiving vehicle; GIso - Ovx and treated with soy extract (150 mg/kg per day); GE2 - Ovx and treated with E2 (10 µg/kg per day). The drugs and vehicle (0.2 mL propylene glycol) were administered for 30 consecutive days after ovariectomy. On the last day the animals were anesthetized, the hearts removed, submerged in 10% formaldehyde and fragments of the ventricles underwent histological procedures, and the sections were stained with hematoxylin and eosin or picrosirius-red. Histomorphometric analysis (number and volume of nuclei and quantification of collagen) was performed under a light microscope with AxioVision Rel. 4.2 software, and collagen fibers were quantified using IMAGELAB-2000 software. Data were submitted to ANOVA followed by the Tukey test (p<0.05). RESULTS: We observed a higher number of cardiomyocyte nuclei in animals of the Ovx and Iso groups than in GE2 and GCtrl animals (GOvx=121.7±20.2=GIso=92.8±15.4>GE2=70.5±14,8=GCtrl=66.3±9.6; p <0.05), while the nuclear volume was greater in the Ctrl and E2 groups (GE2=35.7±4.8 GCtrl=29.9±3.6=>GIso=26.5±4.5=GOvx=22.4±2.9; p <0.05). Collagen concentration was higher in the Ovx group (GOvx=5.4±0.1>GCtrl=4.0±0.1=GIso=4.4±0.08=GE2=4.3±0.5; p <0.05). CONCLUSIONS: Estrogen may prevent the reduction of the nuclear volume of cardiomyocytes and collagen deposition between heart muscle fibers, while the administration of isoflavones only prevents the deposition of collagen, which can preserve the mechanical properties of cardiac fibers.

Efeito da ipriflavona sobre ratas Wistar e suas ninhadas / Effect of ipriflavone on Wistar rats and their litters

OBJETIVO: Avaliar os efeitos da ipriflavona durante a fetogênese, já que não foram encontrados estudos visando avaliar seu efeito durante este período. MÉTODOS: Foram utilizadas 60 ratas prenhes, distribuídas aleatoriamente em quatro grupos (n=15). G-controle (1 mL de água destilada) e três grupos tratados com ipriflavona, via intragástrica, do 16º ao 20º dia pós-coito (PC): G-300 (300 mg/kg), G-1500 (1.500 mg/kg) e G-3000 (3.000 mg/kg). Os animais foram pesados e sacrificados no 21º dia por exsanguinação total sob anestesia (xilazina (10 mg/kg) e quetamina (90 mg/kg) via intraperitoneal. Foi realizado hemograma completo e dosagens séricas de colesterol, triglicérides, AST, ALT, ureia, creatinina e glicose das ratas prenhes. Após laparotomia foram removidos e pesados fígado, rim, suprarrenais, baço e ovários; os fetos e placentas foram pesados obtendo-se o peso médio das ninhadas. Quatro fetos (dois machos e duas fêmeas) por mãe foram aleatoriamente designados para obter-se o comprimento e peso de cérebro, fígado, rins e pulmões. Para a análise estatística utilizou-se o teste ANOVA seguido do teste de Dunnet; para dados não homocedásticos e sem distribuição normal, foi usado o teste de Kruskal-Wallis, seguido de Mann-Whitney; as proporções foram analisadas pelo teste do χ² (p<0,05) RESULTADOS: Níveis de triglicérides (mg/dL): G-Controle (138,8±21,8); G-300 (211,2±63,9); G-1500 (251,5±65,2); G-3000 (217,7±49,6); p<0.05. Peso corporal dos fetos (g): G-Controle (machos 3,3±0,3; fêmeas 3,1±0,3); G-300 (machos 3,4±0,2; fêmeas 3,1±0,4); G-1500 (machos 3,5±0,3; fêmeas 3,2±0,3); G-3000 (machos 3,4±0,5; fêmeas 3,1±0,4). CONCLUSÃO: A ipriflavona não causou toxicidade materna, mas elevou níveis de triglicérides e reduziu o hematócrito em doses elevadas, o tamanho, peso corporal e de órgãos fetais não foram alterados. Não foram observadas malformações externas nem mortes fetais.

PURPOSE: Evaluate the effects of ipriflavone during fetogenesis, since no studies have been conducted to assess its effect during this period. METHODS: 60 pregnant rats were divided randomly into four groups (n=15). G-control (1 mL of distilled water) and three groups treated intragastrically with ipriflavone from the 16th to the 20th post coitus (PC) day: G-300 (300 mg/kg), G-1,500 (1,500 mg/kg) and G-3,000 (3,000 mg/kg). The animals were weighed, anaesthetized intraperitoneally with xylazine and ketamine at doses of 180 mg/kg and 10 mg/kg, respectively, and sacrificed by total exsanguination on the 21st day. A complete blood count was performed and serum cholesterol, triglycerides, AST, ALT, urea, creatinine, and glucose were determined in pregnant rats. After laparotomy, the liver, kidneys, adrenals, spleen and ovaries were removed and weighed; fetuses and placentas were also weighed to obtain the average weight of the litters. Four fetuses (two males and two females) were chosen at random for the determination of the length and weight of brain, liver, kidneys and lungs. Statistical analysis: ANOVA followed by Dunnett's test. For raw data without normal distribution and homoscedasticity, we used the Kruskal-Wallis test followed by the Mann-Whitney test. Proportions were analyzed by the χ² test (p<0.05). RESULTS: Triglyceride levels (mg/dL) were: Control-G (138.8±21.8), G-300 (211.2±63.9) G-1,500 (251.5±65.2) G-3,000 (217.7±49.6); p<0.05. The body weight of fetuses (g) was: G-Control (male 3.3±0.3; female 3.1±0.3), G-300 (male 3.4±0.2; female 3.1±0.4), G-1,500 (male 3.5±0.3; female 3.2±0.3), G-3,000 (male 3.4±0.5; female 3.1±0.4). CONCLUSION: Ipriflavone did not cause maternal toxicity, but increased triglyceride levels and reduced hematocrit at higher doses. The body and organ weights of the fetuses did not change with dam treatment. There were no external malformations or fetal deaths.

Efeitos das isoflavonas em altas doses sobre o útero da rata / Effects of high-dose isoflavones on rat uterus

OBJETIVO: Avaliar o efeito de altas doses de isoflavonas no útero de ratas adultas castradas. MÉTODOS: Ratas virgens ovariectomizadas (n = 40) foram tratadas por 30 dias consecutivos com veículo (GCtrl) ou genisteína nas concentrações 42 (GES42), 125 (GES125) e 250 (GES250) µg/g de peso corporal ao dia. O extrato de soja e o veículo (propilenoglicol) foram administrados por gavagem. Ao final do experimento, foi realizada dosagem sérica de 17 β-estradiol e progesterona, avaliou-se o peso dos animais e dos úteros e foi feito exame colpocitológico. Fragmentos do terço médio dos cornos uterinos foram fixados em formol a 10 por cento e processados para inclusão em parafina para estudo histológico. Cortes de 5 µm de espessura foram corados pelo HE e destinados a estudo em microscopia de luz. Analisou-se a histomorfologia do endométrio, área endometrial, número e área ocupada pelas glândulas, assim como a concentração de eosinófilos presentes na lâmina própria. Os dados numéricos obtidos foram submetidos à análise de variância complementada pelo teste de Tukey-Kramer (p < 0,05). RESULTADOS: Verificou-se que o peso do útero, as áreas endometrial e glandular e o número de glândulas e de eosinófilos foram maiores nos animais dos grupos GES250 > GES125 do que nos outros grupos (GES250 > GES125 > GES42 = GCtrl; p < 0,05). Os dados morfológicos mostraram proliferação endometrial nos grupos ES125 e ES250, que apresentavam endométrio mais desenvolvido que outros grupos. Em todos os animais do grupo ES250 notou-se a presença de metaplasia escamosa. CONCLUSÃO: A administração de isoflavonas em altas doses promove metaplasia escamosa no endométrio.

OBJECTIVE: To evaluate the effects of high-dose isoflavones on the uterus of castrated adult rats. METHODS: Adult, ovariectomized virgin rats (n = 40) were treated by gavage during 30 consecutive days with vehicle (propylene glycol, group GCtrl) or different doses of genistein: 42 (group GES42), 125 (GES125), or 250 (GES250) µg/g body weight per day. Animals were killed, weighed, vaginal and uterine samples were taken for cytologic evaluation, and serum levels of 17 β-estradiol and progesterone were determined. The middle third of the uterine horns was dissected, fixed in 10 percent formaldehyde and processed for paraffin inclusion; 5-µm thick sections were obtained and stained with HE for further histological study under light microscopy. The endometrial morphology and area, number and area of glands, and number of eosinophils in the lamina propria were analyzed. ANOVA and the Tukey-Kramer test were used for statistical analyses. RESULTS: Uterine weight, endometrial glandular area, and number of glands and eosinophils were all higher in GES250 > G125 than in the other groups (GES250 > GES125 > GES42 = GCtrl; p < 0.05). Morphological data showed signs of endometrial proliferation upon treatment with genistein, especially in animals in GES125 and GES250 compared to other groups. In all animals in GES250, signs of uterine squamous metaplasia were observed. CONCLUSION: A short treatment period with high daily doses of isoflavones can promote endometrial squamous metaplasia in ovariectomized rats.

Evaluation of effect of isoflavone on thyroid economy & autoimmunity in oophorectomised women: A randomised, double-blind, placebo-controlled trial.

Background and objectives: The potential of soy isoflavones to interfere with thyroid function has been reported. However, there are limited data regarding their effect on thyroid function and autoimmunity in surgical menopausal women. The present study aimed to evaluate the effect of isoflavones on thyroid function and autoimmunity, menopausal symptoms, serum follicle stimulating hormone (FSH) and estradiol levels in oophorectomised women. Methods: A randomized, double blind, placebo-controlled trial was conducted in 43 oophorectomised women to evaluate the effect of soy isoflavones (75 mg/day for 12 wk) on serum thyroid profile (free T3 , free T4 , TSH, TBG and anti-TPO antibody titres) assessed at baseline, 6 and 12 wk after randomization. Assessment was also done for menopause symptom score (MSS) three weekly, and FSH and estradiol levels at baseline and at study completion. Results: There was a significant alteration in free T 3 levels in the group receiving isoflavones (4.05 ± 0.36, 4.12 ± 0.69 and 3.76 ± 0.55 pmol/l at baseline, 6 and 12 wk, respectively; P=0.02). However, the mean change in various thyroid parameters at 12 wk from baseline was not significantly different between the two groups. MSS was also significantly decreased at 9 and 12 wk from baseline with isoflavones (12.47 ± 8.15, 9.35 ± 5.23 and 9 ± 5.14 at baseline, 9 and 12 wk respectively; P=0.004) with significant improvement in urogenital symptoms compared to placebo. Isoflavones did not significantly affect other parameters during study period. There were no serious adverse events reported and the proportion of patients experiencing adverse events was similar between the two groups. Interpretation and conclusions: Modest reduction in serum free T3 levels in the isoflavone group in the absence of any effect on other thyroid parameters might be considered clinically unimportant.