That work had the purpose to obtain some stable and productive cell suspensions (shake flash cultures) in a reduced volume (300ml) and also the existent differences establishment between the grapevine varieties from the point of view of cellular increasing in the liquid culture medium. Cell suspension cultured in the Murashige-Skoog culture media supplied with 1-naphthaleneacetic acid (2 mg.l(-1)) and kinetin (0,5 mg.l(-1)) it were obtained from callus cultures of tissue internode explants. The measures realized from 3 in 3 days beginning with the 6th day of culture and ending with the 36th day of culture. To all the varieties studied, in our experimental conditions, the cell suspension increasing stopped after 30th days of culture. The rate of increase of the cell culture in suspension registered a maximum medium value to Merlot species (275,10%), superior value to the others species, that being followed by Pinot Noir variety (269,22%), Oporto (251,65%), Negru Tinctorial (247,69%), Cabernet Sauvignon (237,49%) and Burgund Mare (229,73%). Those results could contribute in the future to the possibility of modern techniques usage of cell suspension as alternative method of multiplying in vitro and also in obtaining some secondary metabolites synthetized at cellular level.

The phytocompound genistein, a tyrosin protein kinase inhibitor, the aglicon of the heteroside genistin, represents the main compound in the soybean, Glycine max (Fam. Fabaceae). Recent studies have attributed an anticancer potential to this compound due to its pro apoptotic and anti-proliferative effects. Several papers have demonstrated that genistein is an activator of caspase 3, an effector of apoptosis on different cell lines, including murine melanoma cell lines. Few data exist however about the effect of genistein on the initiator caspase 2 expressions on different cell lines. The aim of this study was to determine if B164A5 murine melanoma cell line and its high metastatic derivate B16F10 express caspase 2, an initiator of apoptosis after exposure to different concentrations of genistein. Results have shown that, after exposure to concentrations of genistein within the range 1-150 mu M after 72 h of incubation, B164A5 cell line and its high metastatic derivative B16F10 do not express caspase 2. This result leads to the conclusion that genistein induced apoptosis is not mediated through caspase 2 signalling pathway in case of B16 murine melanoma cell lines.

Camellia seed oil is well known for its industrial uses. However, despite the increasing use of this oil, few studies have investigated which factors impact seed oil production. Thus, the aims of this study are to identify which factors influence the percentage seed oil of Camellia reticulata, a by investigating both seed and environmental variables. Firstly, fruit traits were studied and correlated against percentage seed oil data, and then environmental variables linked to seed oil production were investigated. The first experiments analyzed how well fruit characteristics (size, length, weight, seed number, seed oil) of the Camellia plants predict percentage seed oil. The second experiment modelled the effect of environmental variables (soil, elevation, temperature and rainfall) on seed oil production. The results indicated that the kernel ratio per fruit, seed weight to fruit weight ratio and fruit weight positively influenced percentage seed oil while, when these previous effects are accounted for, the number of seeds reduced percentage seed oil. The environmental model showed that seed oil was influenced most strongly by elevation and soil type, with haplic Acrisols providing the highest seed oil. Thus it is clear that Camellia seed oil is affected by a variety of factors, and these should be taken into account when selecting species and cultivars for the planting of Camellia stands for oil production.

Vegetable oils are a class of natural products very frequently used in phytocosmetology. Natural remedies with antiaging action are very popular in our days. Harmfulness of natural compounds on the skin, is quickly observed by the 24-48 hour patch test skin irritation and erythema measurement. Changes in the concentration of melanin and sebum in the dermis represent real parameters which give information regarding the status of skin aging. These parameters were measured with the device Mexameter MX18 and Sebumeter MS 815. Peanut oil and olive oil induced a slight increase in levels of melanin, during the 28 days of study. Grape seed oil had a mean constant action. Argan oil and sesame induced a slight reduction in the concentration of melanin. It was also noted that the application of peanut oil and olive induced an increase in concentration in sebum. Argan oil, grapes oil, and to a lesser extent, sesame oil induced a lower level of sebum in the dermis.

The purpose of the study was to establish existent correlations between anthocyanins quantity synthetized in the callus culture and the period of culturing at grape vine (Vitis vinifera L.). To obtain the callus cultures it were used four types of explants of those six species of grape vine taken in study. Measures realized after 6,12,18,24,30,36 days of culture of the callus derived from every type of explant. The results obtained showed that highest values concerning the anthocyanins synthesis during the callus culture were registered in the tissue of internode callus (12,50 mg.g(-1)) to species Negru Tinctorial after 24 days of culture and the most reduced values registered in the callus with foliar tissue (0,34 mg.g(-1)) to Cabernet Sauvignon, after 12 days of culturing. Those results could contribute in the future as possibility of cells in vitro cultures usage as alternative source of anthocyanins, natural pigments that presented a large applicability in alimentary, cosmetics, pharmaceutic industry.

Introduction: Soy, Glycine max, Fam. Fabaceae is a species of vegetables originary from East Asia. The vegetal product used both in the pharmaceutical and nutritional area are the beans, Sojae semen. Vegetable soybean is rich in phytochemicals beneficial to the human being and is therefore considered a nutraceutical or a functional food crop. The aim of this study is to test the antiproliferative effect of soy total extract, employing different concentrations, on B164A5 murinic melanoma cell line, using the MTT proliferation assay. Material and method: Soybean seeds were grounded and a solvent formed of DMSO-ethanol-water in 5-70-25 v/v/v ratio was prepared. The extraction was made at room temperature using an ultrasonic bath (Falc LCD Series) for 30 minutes, 59 kHz. The solvent was evaporated with a rotary evaporator at 50oC. B164A5 cells were incubated 24 h with 100 μM, 50 μM, 30 μM, 15 μM, 1 μM, 0 μM soy total extract. Results: MTT analysis showed an inhibition of all chosen concentrations of soy total extract. The inhibition was directly proportional with the concentration of soy total extract. Additionally, a change in the number and morphology of B164A5 cells was noticed starting from the concentration of 15 μM. Conclusion: Our results suggest that soy total extract presents an antiproliferative effect in vitro on B164A5 murinic melanoma cell line.

Background The isoflavonoid genistein represents the major active compound from soybean, the vegetal product from Glycine max (Fabaceae) . The aim of this study is to prove that genistein was incorporated in two semisynthetic cyclodextrins, beta-cyclodextrin derivatives: hydroxypropyl-beta-cyclodextrin and randomly-methylated-beta-cyclodextrin as well as to compare the anti-inflammatory activity of genistein with that of genistein incorporated in these two types of semisynthetic cyclodextrins. Results The animal studies were conducted on 8-week old C57BL/6 J female mice. Inflammation was induced in both ears of each mouse by topical application of 10 micrograms 12-O-tetradecanoylphorbol-3-acetate dissolved in 0.1 ml solvent (acetone : dimethylsulfoxide in a molar ratio 9:1). Thirty minutes later treatment was applied. The inflammatory reaction was correlated with increased values in ear thickness. Treatment with genistein and genistein incorporated in the two cyclodextrins led to decreased values for ear thickness. Better anti-inflammatory action was found for the complexes of genistein. Both haematoxylin-eosin analysis and CD45 marker expression are in agreement with these findings. Conclusions Results allow concluding that genistein is an active anti-inflammatory phytocompound and its complexation with hydrophilic beta-cyclodextrin derivatives leads to a stronger anti-inflammatory activity.

GEORGETA POP*1, ATENA GALUSCAN2*, CAMELIA PEEV2, ANDREA MILITARU2, LAURIAN VLASE3, LAVINIA ARDELEAN2, LAURA CRISTINA RUSU2 1 Banat‘s University of Agricultural Science and Veterinary Medicine, 119 Calea Aradului, 300645 , Timisoara, Romania. 2 University of Medicine and Pharmacy, “Victor Babes”, 2 Eftimie Murgu, 300041, Timisoara, Romania 3 University of Medicine and Pharmacy “Iuliu Hatieganu”, 8 Victor Babes, 400012, Cluj Napoca, Romania

In this paper we studied the identification and quantification of sterol fractions from extracted oils in different types of rapeseed cultivars. Rapeseed (Brassica napus L.ssp.oleifera) is one of the most used oilseed crop. They serve many other dietary purposes including cooking and salad preparation. The quality of edible oils can be considered from a consumer acceptance. In the characterization of the oil identity composition of sterols fractions are very useful. Sterols are present naturally in small quantities in vegetable oils. Including plant sterols in the diet may lower blood cholesterol levels, being a topical study for researchers in our days. To analyze sterols from vegetable oils we used high performance liquid chromatography coupled with spectrometry mass (HPLC/MS). Analitical column used is Zorbax SB-C18 100 mm x 3.0 mm i.d., 5 mu m, pre-column Zorbax SB-C18. Analysed sterols were: cholesterol, beta-sitosterol and stigmasterol. It was established the minim detection limits, regression coefficients and reproductibiliy of the method. Stigmasterol and cholesterol content in analysed samples was under the quantification limit of the method (under 200 ng/mL), sitosterol content registered was variable between large limits of 1.3-1375.6 mu g/g, depending on the rape cultivars from which the oil was extracted.

Betulin and betulinic acid are pentacyclic triterpenes present in the bark of the birch tree and other vegetal sources. Quantitatively, in birch bark betulin is more significant than betulinic acid; therefore, birch can be a large and feasible source of raw material for betulin extraction. Betulin can be used as extracted or, after chemical modification, as a starting compound for its acid, betulinic acid, with both substances possessing various interesting pharmacological properties. The purpose of this study is to analyse the betulin and betulinic acid content of a birch tree bark extract, as well as its cytotoxic activity. The extraction was done using a Soxhlet extractor and chloroform/dichlormethane/methanol (1 : 1 : 1) as solvent. The betulin and betulinic acid content of the extract was estimated using standards of pure betulin and betulinic acid, by thermal analysis as opposed to pure substance (thermogravimetric and differential thermal analysis). The extract and the main compounds were also analysed by NMR. The results indicated a high amount of betulin in the final extract (up to 50%), and an important quantity of betulinic acid: over 3%. The cytotoxic activity indicated a high proliferation inhibition for the birch tree extract but was still comparable with betulinic acid and betulin.

The aim of this study is to make a comparison between the action of genistein and total soy extract regarding anticancer action on two different in vivo models: phytobiological test and animal model, and to see which of the two tested samples present a greater antiproliferative effect. Soybean seeds were grounded and a solvent formed of DMSO-ethanol-water in rapport 5-70-25 v/v/v was prepared. The extraction was made using an ultrasonic bath (Falc LCD Series) for 30 minutes, 59 kHz. The solvent was evaporated with a rotary evaporator at 50°C. Genistein was acquired from Extrasynthèse (France), hydroxypropyl-γ-cyclodextrin (HPGCD) from Cyclolab Hungary, 7,12-dimethylbenz[a]anthracene (DMBA), dimethylsulfoxide (DMSO), and 12-O-tetradecanoylphorbol-13-acetate (TPA) from Sigma Aldrich, Germany. Because of the poor water solubility, genistein was prepared in a complex with hydroxypropyl-γ-cyclodextrin in a molar ratio 1:2 by kneading method and total soy extract in a mass ratio 1:4 also by kneading method. Phytobiological test indicated an inhibition index over 50% in case of solutions of concentration between 8-33% in both samples, suggesting a possible antiproliferative action at a superior level. Study on C57BL/6J mice was made on which it was induced cancer with physical agents like DMBA, and it was promoted with TPA. Mice where divided in four groups: Group A - blank group, Group B - mice who received total soy extract, Group C - mice who received genistein, Group D - untreated mice. Results on animal model show that both soy total extract and genistein inhibited the initiation and promotion of chemically-induced skin tumorigenesis, but genistein had a greater success in recovering skin lesions type experimental malignant melanoma.

Ochratoxin A (OTA) is a secondary fungal metabolite that enters the food chain by cereals, beer and other products. Its toxicity is an important aim regarding the human pathologies such as nephrotoxicity. This mechanism is intense studied because of the affinity for blood albumins and the renal accumulation by the organic anion transporter. Its serum half-life is different in humans (850 h) and chicken (4.1 h) after oral administration. These data could lead to the idea of analyzing the deep mechanism in contact with blood elements. An important protocol for observation of necrosis/ toxicity and angiogenesis is CAM (chorioallantoic membrane assay) developed on embryonated chicken eggs. This test could be correlated with the red blood cell test (RBC). In this study the toxicological effect of Ochratoxin A was tested. The Ochratoxin A was disolved in corn oil, in the similar concentration used in test on rats. The lipophilic solvent assures an important penetrability for tested compound on vascular plexus. The evolution of embryo vessels was observed after 15 minutes, 1h and 1 day. Samples were collected for haematoxilin-eosin staining and imunohistochemical evaluation. The same corn oil solution was used for the tests on blood red cells to see the damages. The OTA was also administered to Sprague Dawley male rats and a detailed blood test was made. The main results indicated that OTA influences the blood vessels and blood quality in vitro and in vivo . The irritation created on blood vessels is moderate comparing to strong irritants but it is significant. It determines moderate changes on blood elements after a period of presence of a few weeks in sytemic ic circulation.

Betulinic acid (3 beta, hydroxy-lup-20(29)-en-28-oic acid, BA) is a pentacyclic triterpene previously tested on several cell lines of neuroectodermal origin and on some in vivo tumor models, such as the melanoma model. The aim of the present work was to test its activity by increasing its water soluble fraction with gamma type cyclodextrins (hydroxipropilgamma cyclodextrin). The study consisted in a preliminary in vitro evaluation on A431 and MCF-7 cell lines and in vivo tests on fertilized eggs. The dissolution capacity in presence of cyclodextrin was analysed by the phase diagram. The main conclusion was that betulinic acid possessed an important antiangiogenic activity and a significant cytotoxicity, especially on the A431 cell line.

Isoflavonoid genistein (4',5,7-trihydroxyisoflavone), the aglicon of heteroside genistin, represent the major active compound from soybean. It is solubile in organic solvents such as DMSO , dimethyl formamide, acetone, ethanol. Due to its chemical structure it shows, however, poor solubility in water, that of course drastically reduces its bioavailability. The aim of this study is to demonstrate that genistein can be incorporated in different tyes of ramified cyclodextrins, compounds that increase water solubility: hydroxyl-propyl-beta-cyclodextrin (HPBCD) , randomly -metylated- beta-cyclodextrin (RAMEB) and 6-O -Maltosil- beta-cyclodextrin (G 2BCD) . The scanning electron microscopy images show a difference between the structure of the pure substance, genistein and the structure of genistein after the kneading with the three cyclodextrins. Another analyze that was made in order to prove that complexation took place was the differencial scanning calorimetry. Genistein has an endothermic peak which reflect its melting point around 300 oC. HPBCD , RAMEB and G 2BCD are amorphous materials, in these cases complexation phenomena is presumable, because the melting point disappeared. Presented data suggest that incorporation of genistein in hydroxy-propyl- beta-cyclodextrin , randomly -metylated- beta-cyclodextrin and 6-O-maltosil- Genistein Isoflavonoid (4 ',5,7-trihydroxyisoflavone), aglicon de heteroside genistin, reprezinta compusul activ principal din soia. El este solubil in solvenţi organici, cum ar fi DMSO, dimetil formamida, acetona, etanol. Cu toate acestea, datorita structurii sale chimice, prezinta solubilitate slaba in apa, ceea ce desigur ca ii reduce in mod drastic biodisponibilitatea. Scopul acestui studiu este de a demonstra ca genisteina poate fi incorporata in diferite tipuri de ciclodextrine ramificate, compusi care cresc solubilitatea in apa: hidroxil-propil-beta-ciclodextrina (HPBCD), aleator-metilata-beta-ciclodextrina (RAMEB) si a 6-O-Maltosil-beta-ciclodextrina (G 2BCD). Imaginile de microscopie electronica de baleiaj arata o diferenţa intre structura substanţei pure, genistein si structura genistein dupa framântare cu cele trei ciclodextrine. O alta analiza care a fost facuta in scopul de a dovedi ca procesul a avut loc a fost calorimetria de scanare differentiala. Genisteina are un vârf endoterm care reflecta punctul de topire in jurul valorii de 300 oC. HPBCD, RAMEB si G 2BCD sunt materiale amorfe; in aceste cazuri, sunt presupuse a exista fenomene de complexare, deoarece punctul de topire a disparut. Datele prezentate sugereaza ca incorporarea de genisteina in hidroxi-propil-beta-ciclodextrina, aleator-metilata-beta-ciclodextrina si 6-O-maltosil-beta-ciclodextrina a avut loc din cauza schimbarilor aparute in proprietaţile fizico-chimice ale compusilor. Cuvinte cheie: genisteina, hidroxi-propil-beta-ciclodextrina, aleator-metilata-beta-ciclodextrina, 6-O-maltosil-beta-ciclodextrina, SEM, DSC

Glycine max (L.) Merr. Sin Glycine hispida (Moench) Maxim, known as soy is a plant from the Fabaceae family known for its nutraceutical properties. The product used in the pharmaceutical and food area is the soybean, Glycine semen, with a composition formed of proteins 35-40%, lipids 15-20%, sugars 15-35%, isoflavones 0,12-0,3 %. The consumption of soy products has many health benefits, including protection against breast cancer, prostate cancer, menopausal symptoms, heart disease and osteoporosis. Many of the health benefits of soy are derived from its isoflavones. The chemical structure of isoflavones is very similar to that of our own estrogen. Therapeutic daily dose of genistein is 1.5 to 4.1 mg / person and genistin from 6.3 to 8.3 mg / person. For this study grains from two varieties (B and H) of Glycine max (L.) Merr. were considered for quantitative analysis of total lipids (Soxhlet), proteins (Kjeldahl), polyphenols (Folin Ciocâlteu) and isoflavones, daidzin, genistin, daidzein and genistein (HPLC). Conducted tests show that different varieties of soybean have a different quantitative chemical composition in isoflavones. The concentration of total lipids and proteins fit in the values indicated in literature. The polyphenol concentration in the two samples was similar, 313 and 303 mg/100g. The glycosidic structures of isofavones were present in the ethilic alcoholic solution 90%. H variety has higher concentrations of daidzin 819.0 mg /g and genistin 905.6 mg /g towards B variety 460,48 μg/g daidzin and 364,60 μg/g genistin. H variety is preferred to be used in furtherer studies.

Melanoma and lupeol are two aspect that could have a common knowledge because of the activity of pentacyclic triterpene in the mentioned pathology. It has also been shown that lupeol induces differentiation of mouse melanoma cells. The aim of present study was an evaluation of topical active compound lupeol on an experimental chemical melanoma and an in vitro evaluation on sensitive cells such as HeLa (cervix carcinoma) and A431 (skin carcinoma). The methods used specific cells and animals like mice C57BL/6J. The measurements and evaluation included histopathological aspect and erythema evaluation using a Mexameter MX18. The main results indicated an important antitumor activity of lupeol both in vivo and in vitro.

Betulin and betulinic acid are pentacyclic triterpens with antitumor activity, especially on melanoma and skin cancer. Considering their low water solubility, studies have been conducted in order to improve this aspect. One possible solution is the preparation of inclusion complexes with semisynthetic cyclodextrins using different methods. The purpose of this study was the evaluation of the stability constants of betulin and betulinic acid complexes with two cyclodextrins: hydroxypropyl-beta-cyclodextrin and randomly methylated-beta-cyclodextrin, using the Higuchi and Connors phase solubility method. Products have been analyzed by scanning electron microscopy and in vitro tests. The presence of cyclodextrins increases water solubility and biological activity without toxic activity. Rezumat Acidul betulinic şi betulina sunt triterpene pentaciclice cu activitate antitumoralǎ mai ales în melanom şi cancerul de piele. Solubilitatea lor în apǎ fiind foarte scǎzutǎ, s-au realizat studii pentru remedierea acestui aspect. O posibilǎ abordare este prepararea unor complecşi de incluziune cu ciclodextrine, utilizând diferite metode. Scopul acestui studiu este evaluarea constantelor de stabilitate ale complecşilor acidului betulinic şi betulinei cu douǎ ciclodextrine: hidroxipropil-betaciclodextrina şi betaciclodextrina metilatǎ aleator, utilizând metoda solubilitǎţii de fazǎ introdusǎ de Higuchi şi Connors. Produşii au fost analizaţi prin microscopie de scanare electronicǎ şi teste in vitro. Prezenţa ciclodextrinelor conduce la creşterea solubilitǎţii în apǎ şi a activitǎţii biologice, fǎrǎ a accentua toxicitatea acestor compuşi.