Polyphenol-rich extract from Pinus sylvestris L. bark--chemical and antitumor studies.

UNLABELLED: Pinus sylvestris L. is a very important timber tree in Romania, the bark being the main waste from the wood processing. AIM: The aim of this study was to investigate the chemical composition and antitumor effects against HeLa cells of a polyphenol-rich extract from Pinus sylvestris L. bark. MATERIAL AND METHODS: The polyphenolic content and profile were analyzed by means of spectrophotometry and RP-HPLC-UV/ESI-MS in the negative ion mode. The antitumor activity was investigated using two in vitro assays: MTT and flow cytometric apoptosis assays. RESULTS: Pine bark extract contained a high total phenolic content (48.16 +/- 0.29%). RP-HPLC-UV/ESI-MS analysis allowed the identification of taxifolin, a taxifolin-hexoside and several procyanidins (two monomers, three dimers and three trimers). At 200 microg/mL, pine bark extract exhibited a high cytotoxicity against HeLa cells (82.10 +/- 1.95%). Flow cytometry revealed the ability of pine bark extract to induce apoptosis: 71.97 +/- 0.96% apoptotic cells were determined in HeLa cells after a 48 h incubation with pine bark extract (200 microg/mL). CONCLUSIONS: The ability of pine bark extract to reduce viability and induce apoptosis in HeLa cells suggests the presence of compounds with antitumor properties and encourages further studies for their isolation and characterization.

Preclinical trial of the antitumoral therapeutic effectiveness of some natural polyphenolic biopreparations.

We have assessed the antitumoral action of the POLYAS I and POLYAS II vegetal polyphenolic biopreparations--separated and purified from Asclepias syriaca leaves - in rats with various experimental tumoral lines. We studied the therapeutic effect of different doses on the tumor generation process and compared it with the experimental oncostatic action of several standard chemotherapeutic drugs of clinical use (thiotepa, methotrexate, melphalan and cyclophosphamide). In our experimental treatment with the bioactive polyphenolic agents, we have used various doses, both higher and lower than the dose that had conditioned the expression of their antitumoral action upon Guerin T-8 lymphotropic epithelioma and upon Walker 256 carcinosarcoma. We found the antineoplastic effectiveness of those aromatic biopreparations from phytomass to be dose-dependent. We compared the evaluation indices of the antitumoral pharmacodynamic effect we obtained in the treatment with the POLYAS biopreparations with those of reference cytostatic agents. The antitumoral potential of the new natural biopreparations is higher than, equal or close to that of the standard oncochemotherapeutic agents. Antitumoral effectiveness can be improved by an experimental manipulation of the therapeutic doses--which proves the existence of a dose-response relationship. POLYAS I and POLYAS II polyphenolic biopreparations are compatible in point of effectiveness with the standard cytostatic agents, a fact that we considered relevant for the characterization of the POLYAS I and POLYAS II vegetal extracts as potential antineoplastic agents. The quantitative preclinical evaluation of the specific pharmacodynamic effect will be complemented by the investigation of the new polyphenolic biopreparations therapeutic effectiveness in tumors with various degrees of development.

Preclinical qualitative evaluation of the antitumoral pharmacodynamic action of some natural polyphenolic biopreparations.

We have investigated the impact of POLYAS I and POLYAS II polyphenolic biopreparations - specifically separated and purified from Asclepias syriaca leaves, and characterized in vitro as cytotoxic and/or cytostatic agents - on the tumor generation process. A series of in vivo tests of their effect on the development of Guerin T-8 lymphotropic epithelioma and Walker 256 carcinosarcoma were conducted. In a first stage of preclinical trial we had used several tests meant to evaluate their antitumoural activity indices. The same tests were then used under similar experimental conditions in the solid tumoral systems mentioned. A comparative analysis of the antitumoral activity evaluation indices resulting from our tests with the reference indices set by the American and German preclinical screening programs pointed to their compatibility. Thus, we found similar values of mean tumoral regressions, of the ratio between mean tumoral weights of the treated and control groups, respectively, of the T/C products resulting from successive re-tests. Also T/C values resulting from retests were within the limits of admissible variability range. All those results highlighted the antineoplastic pharmacotherapeutic effect of the polyphenolic biopreparations and also proved that effect to be replicable. The qualitative evaluation of the pharmacodynamic action of those preparations was a condition for their further quantitative pharmacological evaluation in point of antitumoral therapeutic effectiveness in a preclinical stage.

Reactivity of HeLa tumoral cells under the in vitro action of some aromatic compounds from the phytomass.

The in vitro action of some natural polyphenolic preparations, extracted from the leaves of Asclepias syriaca, upon the proteinosynthesis of HeLa cancerous cells and, implicitly, upon the development of HeLa cells cultures was investigated. The significant perturbation of proteic biogenesis, the inhibition of HeLa tumoral cells cultures development, as well as the existence of a dose--response relationship argue the behaviour of these products as in vitro active cytostatic agents. This characterization justifies their introduction in the in vivo screening program on rats bearing of different experimental tumoral lines, for the preclinical pharmacological evaluation of the POLYAS I and POLYAS II vegetable polyphenols antineoplastic activity.

Feeding behaviour in rats with isolated medial hypothalamus as a function of ambient temperature.

The experiments of mechanical isolation of medial hypothalamus from the lateral hypothalamus and the preoptic anterior hypothalamic (POAH) region in rats showed that: 1. The interruption of neural connections between POAH area and medial hypothalamus do not prevent the decrease of food intake which normally occur in a hot environment. 2. At 33 degrees C, hyperphagic rats gained more weight than sham-operated ones. 3. At 4 degrees C, rats made hyperphagic by hypothalamic isolation do not ajust their food intake for a long period and do not gain weight. 4. The excitatory pathways of the feeding center from the POAH area do not penetrate directly into the lateral hypothalamus, but rather into the medial retrochiasmatic area. 5. The temperature influences the diurnal pattern of feeding only in rats with intact or unilateral neural connections of the hypothalamic structures 6. It seems that the thermostatic mechanism, which is a potent regulator of feeding, is closely associated with the central control of thyrotropin release, and that the hypothalamic structures may be considered only as a necessary link in the nervous mechanism involved in feeding control.

The effect of partial or total medial hypothalamic isolations on lipid metabolism in rats.

Medial hypothalamic isolation in the rat occurring under free food acces or after 24 hours starvation determines a significant increase in serum lipemia, serum esterified fatty acids and carcass fat. A significant rise in carcass fat was evident in rats made hyperphagic by medial hypothalamus isolation and maintained on limited food intake. It has been concluded that the VMH nuclei are involved in the control of the lipid metabolism primarily through the inhibitory effect exerted on the lateral hypothalamic parasympathetic area.

Lipid metabolism in rats with isolated medial hypothalamus.

Based on determinations of the serum total cholesterolemia, triglyceridemia, free fatty acids and obesity Lee index, performed in rats with isolated medial hypothalamus--VMH included--which had either free food access, or after 24 hours of starvation, or maintained on limited food intake, it has been concluded that the VMH nucleus is involved in the control of lipid metabolism especially through its inhibitory action on the lateral hypothalamic parasympathetic area.

Feeding behavior in the rat with isolated medial hypothalamus.

After complete, unilateral, frontolateral, dorsal isolation of the medial hypothalamus, VMH included, or fornix section above the hypothalamus, total food consumption and diurnal pattern of food intake were followed 85 days postoperatively. It is suggested that the saftety signals are generated not only in VMH nucleus, but in a VMH-retrochiasmatic region located anteriorly to the VMH.