Red clover is a perennial forage crop rich in isoflavones, the bioactive compounds with a positive effect on human and animal health. The aim of the research was to determine a variation in the ...isoflavone contents in the Croatian red clover collection and to identify the most promising materials to be used in the breeding program for the development of new cultivars for specific purposes. Leaf samples of 29 red clover cultivars/populations (two cultivars, twenty breeding populations, and seven local populations) were collected in a full flowering stage, and the identification and quantification of isoflavones was performed using the HPLC analysis. The most common isoflavones in the red clover cultivars/populations were formononetin and biochanin A. A significant variation among the cultivars/populations in the total and individual isoflavone content was determined. The populations with very high and low contents of both the total and of the individual isoflavones were identified to be used for the breeding purposes in order to develop new forage cultivars, or for specific goals in the pharmaceutical industry.
Crvena djetelina višegodišnja je krmna kultura bogata izoflavonima, bioaktivnim spojevima s pozitivnim utjecajem na zdravlje ljudi i životinja. Cilj istraživanja bio je utvrditi varijabilnost sadržaja izoflavona hrvatske kolekcije crvene djeteline te identificirati najzanimljivije materijale koji će se koristiti u oplemenjivačkome programu za razvoj novih sorata za specifične namjene. Uzorci lista prikupljeni su u stadiju pune cvatnje iz 29 sorata/populacija crvene djeteline (dvije sorte, dvadeset oplemenjivačkih populacija, sedam lokalnih populacija), a identifikacija i kvantifikacija izoflavona obavljena je pomoću HPLC analize. Najzastupljeniji izoflavoni u sortama/populacijama crvene djeteline bili su formononetin i biohanin A. Utvrđena je značajna varijabilnost među sortama/populacijama u sadržaju ukupnih i individualnih izoflavona. Identificirane su populacije s vrlo visokim i niskim sadržajem kako ukupnih, tako i pojedinačnih izoflavona koje će se koristiti u oplemenjivačke svrhe za razvoj novih krmnih kultivara ili za specifične namjene u farmaceutskoj industriji.
Andropause is defined as a gradual decline of serum testosterone concentration during ageing, which is related to an increased incidence of cardiovascular diseases, benign and malignant prostate ...diseases and osteoporosis. Using steroid hormones, as well as vitamin D and calcium in the treatment of osteoporosis and other ageing symptoms, in both genders, have some undesirable side effects, such as increased risk of cardiovascular diseases, hyperphosphaturia and prostate cancer. Taking into account the potentially harmful aspects of the hormone replacement therapy, an increasing emphasis is placed on the alternative, plant-originated therapeutics for osteoporosis. Accumulating evidence suggests that soy isoflavones may represent a promising alternative remedy for aging symptoms in both genders. This study aimed to examine the effects of steroids, isoflavones and calcium on the structural and functional changes in parathyroid glands and specific functional proteins in the kidney tubules, responsible for Ca and Pi regulation, in an animal model of the andropause, and to determine their potential impact in the preservation of mineral metabolism impaired with ageing. Fifthteen-month-old Wistar rats were orchidectomised (Orx) or sham (SO) operated under ketamine anesthesia. After 2 weeks of recovery, Orx animals were divided into experimental groups and treated subcutaneously with testosterone-propionate (5 mg/kg b.w.; TP), estradiol-dipropionate (0.625 mg/kg b.w.; EDP), vitamine D (50 μg/kg b.w.; Vit D), genistein (30 mg/kg b.w.; G), daidzein (30 mg/kg b.w.; D) or calcium intramuscularly (28.55 mg/kg b.w.; Ca) every day, for 3 weeks. Every treated group had a coresponding SO and Orx control groups treated with the proper amount of vehicle, following the same regime. Animals were fed a soy-free diet with corn oil as the fat source. The rats were decapitated 24h after the last injection, parathyroid glands and left kidneys were excised, their weight was measured and they was processed for light and electron microscopic examinations. Techniques of histochemical staining and immunohistochemical labeling, transmission electron microscopy, and stereological analyses were performed. Gene expression levels were determined with Real-time PCR. Concentrations of PTH, Ca2+, Pi and creatinin levels in serum and concentrations of Ca2+, Pi and creatinin in urine were determined biochemically. The obtained data were statistically processed. The volume of parathyroid glands in Orx rats was increased compared to the SO group. After treatments with TP, EDP or Vit D the parathyroid glands volumes were decreased, when compared to Orx animals. The volume density of chief cells after treatment with EDP was decresed, while the volume density of interstitium was increased, comparing to Orx rats. Treatment with genistein led to an increase of parathyroid gland volume, while treatment with daidzein decreased the same parameter, with regards to Orx group. The volume densities of chief cells were decreased after treatments with genistein or daidzein, while the volume densities of interstitium were increased, in comparison with Orx animals. Administration of calcium to Orx rats provoked the reduction of parathyroid glands volume, while the volume density of interstitium after same treatment was increased, comparing to the Orx rats. The lack of steroid hormones, provoked by Orx, led to the increment of serum PTH, when compared to the SO control group, while treatments with TP, EDP, Vit D, genistein, dadizein or calcium decreased the mentioned parameter, comparing to the Orx group. NaPi 2a expression in Orx animals was reduced in regards to its abundance in SO animals, although it was increased in TP, Vit D, genistein or daidzein groups, compared to the Orx rats. The treatment with EDP attenuated NaPi 2a expression, in comparison with Orx group. In Orx rats, the staining for PTH1R was stronger when compared to SO group, while the treatments with TP, Vit D, genistein, daidzein or calcium induced reduction of the PTH1R immunofluorescence, compared to Orx animals. The intensity of the PTH1R signal after EDP treatment was slightly reduced in regards to its abundance in Orx group. FGFR and Klotho receptor expression weren’t significantly changed in Orx animals, when compared to the SO controls. After treatment with EDP, the expression of FGFR and Klotho receptors was increased, when compared to the Orx rats. Administration of genistein or daidzein caused the decrease in FGFR expression level, compared to Orx animals. Genistein treatment of Orx rats induced enhancement of Klotho receptor expression level, while daidzein treatment decrease it, in comparison with Orx rats. In Orx animals, Ca2+ and Pi serum concentrations were decreased, while urine Ca2+ and Pi content was increased, in comparison with the SO control. After treatments with TP, EDP, Vit D, genistein, daidzein or calcium serum Ca2+concentrations were increased, when compared to Orx animals. Treatments with TP, genistein, daidzein or calcium induced the increment of Pi serum concentrations, with regards to Orx rats. Aplication of TP, EDP, Vit D, genistein, daidzein or calcium to Orx rats led to decrease Ca2+ urine concentrations, comparing to Orx animals. Urine Pi concentrations after treatment with Vit D, genistein, daidzein or calcium were decreased, while EDP treatment induced enhancement of Pi urine concentration, with regards to Orx rats. Our results showed that the treatments with steroids, isoflavones and calcium of andropausal male rats demonstrate some differences in the level of the same direction effects, demarcating widespread, commonly used therapies and alternative approaches in the regulation of Ca2+ and Pi homoestasis. Application of isoflavones led to the stronger increment in abundance and the expression level of NaPi 2a cotransporter, intensively decreased the presence of PTH1R, abundance and expression of FGFR in the kidney tubules was noticeable reduced, and decreased the concentrations of Ca2+ and Pi in urine, as well as more pronouncely increased the concentrations of Ca2+ and Pi in the serum, when compared to the milder effects of treatments with steroids and calcium. The listed changes may reestablish mineral homeostasis, disturbed by the aging process.
Postepeno smanjenje koncentracije testosterona u cirkulaciji tokom procesa starenja, koje je u čvrstoj vezi sa povećanom učestalošću nastanka kardiovaskularnih oboljenja, benignih i malignih oboljenja prostate i osteoporoze, označava se kao andropauza. Terapije steroidnim hormonima, kao i konvencionalna terapija Vit D i kalcijumom, koje se primenjuju u prevenciji i tretmanu menopauzalnih i andropauzalnih simptoma kod oba pola, ispoljavaju i neželjena dejstava i povećavaju rizik od nastanka kardiovaskulanih oboljenja, hiperfosfaturije, hipertrofije prostate benignog karaktera, kancera dojke i prostate. Zbog brojnih negativnih efekata primenjivanih terapija, u poslednje vreme sve je veći broj istraživanja koja teže pronalaženju rešenja sa ciljem prevazilaženja navedenih problema. Veliku pažnju privlače izoflavoni soje, genistein i daidzein, koji su predmet istraživanja brojnih studija, prvenstveno zbog svog blagotvornog efekta na simptome starenja kod oba pola. Cilj ovog istraživanja bio je rasvetljavanje dejstava steroida, izoflavona i kalcijuma na ključne regulatore homeostaze Ca2+ i Pi u animalnom modelu andropauze, i utvrđivanje njihovog potencijalnog efekta u očuvanju metabolizma minerala koji je narušen tokom procesa starenja. Mužjaci pacova Wistar soja, starosti 15 meseci, su orhidektomisani (Orx) i lažno operisani (SO) u ketaminskoj anestaziji (15 mg/kg b.w.). Dve nedelje nakon operacije Orx životinje su podeljene u eksperimentalne grupe (n=8), kojima su subkutano aplikovani testosteron-propionat (5mg/kg t.m.; TP), estradiol-dipropionat (0.625 mg/kg t.m.; EDP), vitamin D (50 μg/kg t.m.; Vit D), genistein (30 mg/kg t.m.; G), daidzein (30 mg/kg t.m.; D) i intramuskularno kalcijum (28.55 mg/kg t.m.; Ca), svakog dana tokom 3 nedelje. Svaka tretirana grupa je imala svoje kontrolne SO i Orx grupe za poređenje, kojima je aplikovan odgovarajući volumen adekvatnog rastvarača. Tokom izvođenja eksperimenta, životinjama su bile slobodno dostupne voda i laboratorijska hrana, koja nije sadržala sastojke soje ni leguminoza, sa kazeinom kao izvorom proteina. Životinje su dekapitovane 24h nakon poslednjeg tretmana, paraštitaste žlezde i levi bubrezi su izolovani, izmereni i pripremljeni za tehnike svetlosne i elektronske mikroskopije, dok su desni bubrezi zamzrnuti u tečnom azotu i čuvani na -80˚C do korišćenja i izolacije RNK. Analiza tkiva je izvršena korišćenjem svetlosne mikroskopije, tehnike imunohistohemijskog bojenja i metoda stereološke analize, te transmisione elektronske mikroskopije, a ekspresija gena je određena reakcijom lančanog umnožavanja u realnom vremenu. Biohemijskim metodama određene su koncentracije PTH, Ca2+, Pi i kreatinina u serumu, kao i koncentracije Ca2+, Pi i kreatinina u urinu. Dobijeni podaci su statistički obrađeni. Volumen paraštitastih žlezda značajno je povećan kod Orx pacova u odnosu na SO grupu. Kod Orx životinja tretiranih TP-om, EDP-om ili Vit D volumen paraštitastih žlezda je značajno smanjen u poređenju sa Orx pacovima. Volumenska gustina glavnih ćelija je značajno smanjena, dok je volumenska gustina intersticijuma značajno povećana nakon tretmana EDP-om u odnosu na Orx grupu životinja. Tretman genisteinom je uzrokovao značajno povećanje volumena paraštitastih žlezda, dok je tretman daidzeinom značajno smanjio volumen paraštitastih žlezda, u poređenju sa istom vrednošću kod Orx pacova. Tretmani genisteinom ili daidzeinom su statistički značajno smanjili volumensku gustinu glavnih ćelija, u poređenju sa Orx grupom životinja. Nakon tretmana genisteinom i daidzeinom, volumenska gustina intersticijuma je značajno povećana u odnosu na Orx grupu životinja. Kod životinja tretiranih kalcijumom, vo
Različiti proizvodi od soje smatraju se tradicionalnom funkcionalnom hranom za mongolsku populaciju na sjeveroistoku Indije. U ovom je radu ispitan utjecaj dodatka različitih aroma (mango, naranča, ...vanilija i bijela ruža), stupnja inokulacije bakterijama mliječno-kiselog vrenja Lactobacillus rham¬nosus K4E (KX950834) i Lactobacillus helveticus K14 (KU644578) i masenih udjela obranog mlijeka i šećera na senzorne značajke sojinog jogurta. Tijekom deset dana skladištenja u hladnjaku pri 6-8 °C ispitivana su fizikalno-kemijska (pH-vrijednost, titracijska kiselost), mikrobiološka (ukupni broj bakterija te broj koliformnih bakterija, kvasaca i plijesni), organoleptička (aroma, okus, boja, punoća, tekstura i ukupna prihvatljivost) i biofunkcionalna (inhibicija proizvodnje angiotenzin konvertirajućeg enzima, antioksidacijski i antimikrobni učinak, biotransformacija izoflavona) svojstva jogurta. Najbolje je ocijenjen jogurt s aromom bijele ruže, čija je pH-vrijednost tijekom skladištenja bila od 5,65 do 4,20, titracijska kiselost (izražena kao maseni udjel mliječne kiseline) od 0,33 do 0,51 % i ukupni broj bakterija vrsta Lactobacillus od 6.81 do 8,69 log CFU/mL. Inhibicija se angiotenzin konvertirajućeg enzima povećavala tijekom skladištenja s početnih 21,17 % na 81,03 % izmjerenih tijekom petog dana skladištenja, a zatim smanjivala sve do 38,85 % nakon desetog dana. Najveća je antioksidacijska aktivnost zabilježena tijekom petog dana skladištenja (87 %). Sojin jogurt s aromom bijele ruže imao je najizraženiji antimikrobni učinak na bakteriju Listeria monocytogenes, a zatim na bakterije Bacillus subtilis, Staphylococcus aureus, Salmonella typhi i Escherichia coli. Analiza RP-HPLC pokazala je da je u jogurtu nakon 18 sati fermentacije dobiveno 87,3 % aglikona sojinog izoflavona genisteina i 58,4 % daidzeina.
U radu je ispitana antioksidativna i citotoksična aktivnost etanolnih esktrakata mlijeka dobivenog nakon 2 dana fermentacije crne soje pomoću imobiliziranoga soja Rhizopus oligosporus NTU5. Rezultati ...su pokazali da takvo mlijeko ima izraženu sposobnost uklanjanja slobodnih DPPH radikala (76 %). Ekstrakti su mlijeka zatim frakcionirani kromatografijom na silikagelu i izdvojen je nepoznati sastojak, nazvan FBE5-A, izraženog antioksidativnog svojstva. Sposobnost toga sastojka da uklanja slobodne DPPH radikale kudikamo je veća (IC50=7,5 μg/mL) nego vitamina E, koji se obično koristi kao antioksidans (α-tokoferol, IC50=17,4 μg/mL) i približno jednaka vitaminu C (askorbinska kiselina, IC50=7,6 μg/mL). Ispitivanje citotoksičnog učinka FBE5-A pokazalo je da selektivno djeluje na ljudske stanice raka Hep 3B (IC50=150,2 μg/mL), a pritom ne utječe na normalne fibroblaste iz pluća MRC-5 (p<0,05). Rezultati upućuju na to da se mlijeko dobiveno fermentacijom crne soje može upotrijebiti kao funkcionalna hrana i za liječenje raznih bolesti, uključujući i rak.
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