Along the process of development of the product different stages of investigation were sorted at the same time with laboratories studies.
In this opportunity were chosen for its presentation the dates obtained in investigation essays “in vitro” and “in vivo” laboratory in relation to GREEN SAP’ s antitumoral properties because this is its principal therapeutical action.
The scientific studies are being realized in the Scientific and technical Investigations Council (CONICET), Argentinean official institution dependent of Technology and Science Education’s Ministry of the Nation Presidency by its executive unity Botanical and Pharmacologic studies Centre (CEFYBO).
INVESTIGATION STUDY’S PROTOCOL
Scientific studies made with GREEN SAP
Kinetic essays of cellular proliferation
These essays will be made on 2 cellular types:
1. Tumor cells: lymphoid tumor line of murin origin BW 5147 which express H-2k haplotype, CD3+ and TCRaß, the ones are tested usually by flux citometry using specific antibodies against the correspondent surface markers.
2. Normal Lymphocytes T from lymphatic nodes of rats from BALG/c with 3-4 months of age, obtained in a aseptic way.
The essays in vitro will be done in sterility conditions in cultivations of 96 wells in a final volume of 0.2 ml.
The effect on cellular proliferation of the mixture to be essay will be evaluated at different times of cultivation (24, 48, 72 y 96 hs.) and in absence (Control) or presence of different concentrations of mixture so as to determine the kinetic action of the product. The concentrations to be tried will be series of dilutions in half in a rate of concentrations that include the proposed dose to be used in human beings (from 1/250 till 1/4000).
Cellular proliferation will be evaluated with the trytiade tymidin incorporation technical (20 Ci/mmol). For this, the cellular cultivations will be pulsed for a period of 16 hs in the case of normal lymphocytes and of 6 hs for tumor cells with trytiade tymidin, after this cellular nucleus are kept by glass fiber paper filtration, Whatmann GF/A. The radioactive tymidin incorporated to DNA will be counted in a Wallac count of liquid glitter.
The cells concentration to be used will be: for tumor line of 3×105 cells/ml (final optimum cellular concentration) and for normal cells 2×106 cells/ml. Both types will be cultivated in RPMI 1640 supplemented with 10% of bovine fetal plasma and 2mM of glutamine and in presence of penicillin (100 U/ml) and streptomycin (100 µg/ml).
Once it was proved the efficacy of the mother tincture’s formulation by the with animal’s experimentation, it has been made a large specter with different dilutions and vehicles.
From this specter it obtained the results of Table 1: “Curve Dose – Answer”
As it is shown in Figure 1: “Graphic Curve Dose – Answer” the optimum dose (best therapeutic effect with no side effects) is 1/400 and the optimum formulation is PF3, the one showed a percentage of cellular proliferation of inhibition of 13.8 %, at this dose.
Making an extrapolation of the obtained results the 1/400 dilution is the same to a dosage of 240 drops per day. This result is obtained considering the volume of a drop is 0.05 ml., so 240 drops are 12 ml. of Basquade solved in blood (adult’s blood volume = 5.000 ml).
The dosage administered of 12 ml. of Basqüadé is solved in 5.000 ml. of blood, that is the equivalent of 1 ml. in 416 ml of blood.
It’s important to remark that in this study was proved that the product must be shaken before its administration for a good homogenization of it, according to its organoleptic properties.