Concentration (125, 250, 500 and 1000 µg/ml) of healthy and fresh drug supplement were prepared by dissolving dry supplement in saline phosphate-buffered PBS.
Perform an Anti-Migraine Test
In this test, a measure of nitric oxide was used to evaluate the therapeutic effect of live and healthy drug supplement. For this purpose, mouse endothelioma F–2 cell line (Cellular Bank of Japan) was used. Availability, high reproducibility, and adequate knowledge of their growth and reproduction conditions were among the factors influencing the selection of this cell line. These cells were placed in 25 cm2 flasks in DMEM (modified eagle Dulbecco medium) (sigma) with 10% fetal calf serum content (Roche) and 1–2% penicillin-streptomycin (sigma) CO2 was multiplied by 5%. The cells were isolated in subconfluent using thripsin (sigma) at a concentration of 25% from the floor. The cells isolated from the flask floor were immersed in the medium. Then, centrifugation, cell deposition was immersed in the new culture. The cells were then transferred to the new flasks or into the container for testing.
Determine Cell Poisoning
To determine the survival of the cells, the test was carried out using a 3-(4,5-dimethylthiazol–2-yl)–2,5-dipheneyltetrazolium bromide) MTT, which is based on the MTT-related mitochondrial-respiratory resuscitation. By performing this test, the maximum concentration that did not cause cell poisoning was determined (1000 µg/ml), several concentrations (500 250 125 µg/ml) of the less than maximum concentration were tested.
The concentration of 50 µg/ml was the lowest concentration that affected NO, although its effect was not significant.
Measurement of Nitrite
Nitrite concentrations were measured as an indicator of nitric oxide production by Griess method. In this method, the sulfuryl amide and N-(–1-naphthyl) ethylenediamine are added to the sample to measure the concentration of nitrate. Sodium nitrate was also used as standard. U1 100 supernatant medium was mixed with 100 µl grease containing 2% sulfonyl amide in 5% chloride oxide and 0.1% N-(1-naphtyl) ethylenediamine. The optical absorption was then read by ELISA at 540 nm. The higher the concentration of nitrate in the sample, the greater the absorption in this wave, thus, it can be compared with the control method by reducing the grease or increasing the amount of nitric oxide in the samples.
In order to induce seizure, on 25th day after birth, each puppy was injected into control and treatment groups with healthy and live drug supplement in 150 mg/ kg subcutaneously. Pilocarpine was purchased from Sina Drug Company. Following injection, the behavior of each rat was recorded by a digital camera and two observers, and the degree of epileptic seizures in this study was categorized as follows.
Step 1: Immobility
Step 2: Lower the front or tail
Step 3: Repeated movements, head bobbing
Step 4: Stay on two feet and fall
Step 5: Getting up and falling steadily
Step 6: Tonic attacks and severe clonic
Other parameters, such as the duration of the first change in behavior and the duration of maximum seizure, were evaluated. Animals were also observed for 24 hours for lethal side effects of pilocarpine.
Signs of seizure (the six steps mentioned above), the latent time to start epileptic seizures, the time needed for maximum response and death and mortality of the rats up to 24 hours after pilocarpine injection in the control and stress groups were compared by Mann-Whitney test. Non parametric were compared. All results were presented as ± Mean error and p <0.05 was considered as meaningful.
Then the of treatment results of this disease were compared between groups receiving Dapsone at concentrations of 10 and 15 mg / kg and groups receiving healthy and live drug supplement at concentrations of 12.5, 25 and 50 mg / kg.
In order to analysis the data in this study, we first make sure that the distribution of data is normal, using Kolmogorov-Smirnov test. Also, in this study, in order to evaluate the significance of the data, it is recommended to use ANOVA test. ANOVA test was used to investigate the differences between and within groups. P<0.05 indicates statistical significance. In order to clarify this issue, by Scheffe post hoc test, this significance was tested one by one between groups.