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project mutamba aids\ hiv

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  • until December 2014, we have animal testing, testing in glass inhibition gave 100 percent, the prognosis for cure and aproxidamente 98 percent, will present the awards in New York on December 1, 2014
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  • 1. 1. CLIENT'S REGISTRATION DATA NAME: Paulo Antônio Rodrigues Gouveia ADDRESS: Rua Goianesia, nº 78, Conjunto Urbanístico. CITY: Araguaina – TO Zip Code: 77818-772 CNPJ/CPF NUMBER: 388.684.581-87 STATE REGISTRATION NUMBER: --- 2. SAMPLE DATA PRODUCT IDENTIFICATION: Aqueous "extract" of Guazuma ulmifolia. TOTAL SAMPLE: 5 bottles. PLACE OF PRODUCTION: Supplied by the client. EXPIRATION DATE: --- BATCH: --- 3. ASSAYS ASSAYS PERFORMED: Purification of the aqueous "extract" of Guazuma ulmifolia and study of the in vitro antiviral (HIV) activity. 4. ASSAYS CARRIED OUT BY: KYOLAB LABORATÓRIO LTDA. CNPJ: 05.758.608/0001-01 Rua Lauro Vanucci, 1020 – Jd. Santa Cândida – Campinas – SP CEP: 13087-548 PERSON IN CHARGE OF ASSAY: Thaís Barbizan Ferreira da Costa and Dr. Amilcar Tanuri DATE SAMPLE RECEIVED: February/2012 Period assay performed: April/May 2012 Date report issued: 06/01/2012 Kyolab, 01st, June 2012 KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 2. Technical Report Purificationof the aqueous “extract” of Guazuma ulmifolia and study of the in vitro antiviral (HIV) activity. 1. Identification of the sample Aqueous extract of Guazuma ulmifolia supplied by the client. 2. Objectives Purification of the aqueous extract and evaluation of in vitro antiviral (HIV) activity. 3. Method 3.1 Purification of the aqueous extract: The extract provided was filtered then submitted to liquid-liquid partition with hexane and butanol, as shown in flowchart 1. The hexane extracted very little, therefore a significant mass was not obtained. Thin Layer Chromatography was performed for comparison of the chemical composition of the different samples obtained from the partition. The following samples were sent for evaluation of the in vitro antiviral (HIV) activity: Butanol fraction (Bul), Aqueous fraction (AQI) and total extract after filtration (TI). KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 3. Aqueous extract of Guazuma - Separatory funnel; - 200 ml Hexane; - Stirring and phase separation. Hexane Fr Aqueous Fr - 200 ml Butanol; - Stirring; - Phase separation. BuOH Fr 1 Grouping of the two fractions . Aqueous Fr - 100 ml Butanol; - Stirring; - Phase separation. BuOH Fr 2 M = 166,7 mg Aqueous Fr M = 1,4662 mg Flowchart 1: Liquid-liquid partition of aqueous extract of Guazuma ulmifolia. KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 4. Thin layer chromatography: Fractions: 1) Aqueous extract after filtering; 2) Aqueous Fraction; 3) Butanol Fraction; Eluent: Ethyl acetate: Formic Acid: Water (90:5:5) Disclosing solution: Anisaldehyde solution. 3.2 Antiviral action test of the fractions (BUI and AQI) and extract (TI) of Guazuma ulmifolia using MT4 cells. 3.2.1 Standard virus: NL4-3 isolate (subtype B, standard) purified by passage in MT4 cell culture. Title: 103 TCID50 / ml. 3.2.2 Cell line used: MT-4, lymphocyte cell line established in culture, CD4+, expressing co-receptors CCR5 and CXCR4 of HIV-1. Syncytium-inducing (SI) cells and very sensitive to infection by HIV-1. KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 5. 3.2.3 Assay design: Infection in 96-well plate, containing 104 cells/well, infected with MOI (multiplicity of infection) of 0.002 (NIH recommendation = 0.001 to 0.01). The three samples (BUI, TI, and AQI) derived from Guazuma ulmifolia were diluted in DMSO at 20mg/ml and subsequently diluted in RPMI 1640 base medium to 200μg/ml in the first well of cells previously infected with the HIV-1 isolate NL4-3 (subtype B). The cells were then exposed to decreasing concentrations of the drug, based on a dilution factor of 5. The culture medium used was RPMI 1640 with the addition of 10% of foetal bovine serum, 1% antibiotics streptomycin/penicillin and 0.2mg ml L-glutamine. The most concentrated well had a final concentration of 200μg/ml, followed by successive 5-fold dilutions as indicated: 40μg/ml, 8μg/ml, 1.6μg/ml, 0.32μg/ml. The last and ninth well was kept as infection control, without the presence of drug. Each array of 10 infected wells was produced in sextuplicate, for subsequent statistical analysis. A fourth array of 10 wells of cells was exposed to serial dilutions of the drug, as described, but without the presence of virus, for analysis of cytotoxicity of the drug at this concentration range. The infection, kept in a 5% CO2 incubator at 37°C, was monitored daily by optical phase microscopy, for analysis of the appearance of syncytia, which generally occurs on the fourth day post-infection. 3.2.4 Assay disclosure: The Promega CellTiter-Blue Assay staining technique was used to measure cell viability on the sixth day p.i., to enable differentiation of cell viability after syncytium-induction. After staining, the 96-well plate is read in a fluorimeter at 560Ex/590Em. The results were analyzed in a Microsoft Excel matrix for Windows (Office 98), with correction of the blank, and the emission frequency of the assay was plotted on a graph (as a percentage, using as 100% standard the emission of wells with viable cells without infection) to determine cell viability. The value of 50% of the emission of the standard was considered as the cut-off value for the calculation of IC50 (inhibitory concentration of the drug in 50% of infection) after obtaining, on the graph, the equation of the logarithmic (or semi-logarithmic) regression curve. A new experiment with the Bu1 sample was performed, to corroborate the previous data and also to confirm the anti-HIV effect of this sample. KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 6. 4. Results The table below shows the raw data for the assay with the samples of Guazuma ulmifolia. Experiment 1: ug/ml 100 1 3218.536 3742.886 3791.326 3111.216 3035.973 3018.325 20 2 3787.642 3349.466 3862.401 19477.83 11976.43 10846.91 4 3 3054.273 2993.495 3165.824 4366.21 8528.929 24493.36 0.8 4 3029.623 2921.36 3175.966 2674.602 5251.845 10675.08 0.16 5 3050.815 3504.554 3471.753 3761.903 4034.446 10511.61 0.032 6 2942.556 3313.594 3284.441 3076.742 6046.562 11035.92 0.0064 7 2838.061 3368.63 3324.773 6052.958 9489.978 13717.92 0.00128 8 3266.952 3508.849 3071.067 3048.72 7767.46 7460.173 0 9 3856.086 3831.135 4265.1 7240.043 10664.81 11631.7 MOCK 10 21463.08 14058.51 20294.67 7477.002 12772.81 14904.05 T1 1 4678.774 3648.367 4505.607 3478.071 3464.056 3466.57 2 4786.826 4698.749 4855.646 11082.36 18769.24 30047.38 3 3640.615 3396.308 3050.558 20798.67 23575.37 42701.53 4 3197.269 3033.221 3060.215 3092.523 6290.354 20304.38 5 2969.581 2937.966 2851.461 6175.344 11040.51 21610.14 6 3167.663 3177.718 3222.067 9931.44 12574.45 33459.76 7 3161.772 3185.137 3087.069 6314.826 6861.942 23847.6 8 2945.539 2912.907 3152.385 6791.282 10059.75 20340.6 9 2893.992 3171.715 3232.763 7868.27 20465.64 24218.94 10 15247.06 16203.14 19429.31 11966.43 22468.87 29270.19 BU1 1 3780.665 3725.434 3694.26 3706.6 3751.745 3786.029 2 6687.205 9439.628 6138.598 9131.073 13926.65 21043.8 3 4030.617 3754.924 3697.187 9456.156 13667.83 35402.97 4 3735.877 3216.574 3453.84 4017.167 5648.854 5648.854 5 3343.416 3391.877 3268.927 6634.354 28670.71 40455.04 6 3349.616 3571.244 3461.059 4801.022 11885.63 36367.43 7 3335.424 3594.279 3536.97 3718.691 6658.553 14835.89 8 3387.817 3599.979 3485.578 4119.047 3977.721 9229.815 9 3535.693 3483.987 3595.887 19062.17 6637.935 14016.52 10 21099.38 20940.39 28091.88 10836.41 13054.59 23878.32 AQ1 KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 7. Figure 1: Survival of the MT4 cell with different concentrations of samples BU1, TN1, and AQ1. Partial protection of the cells can be seen with the BU1 sample (30%) at the concentration of 20ug/ml. Experiment 2: The table below shows the data for repetition of the experiment with the Bu1 sample. KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 8. Cell Viability (%) BU1 antiviral BU1 cytotox Figure 2: Repetition of the previous experiment with the extract BU1. The histogram shows survival of the MT4 cell infected by the HIV-1 pNL43 virus with a MOI of 0.01 and simultaneously, the cytotoxicity with different concentrations of the BU1 sample. A partial protection of the cells can be seen with the BU1 sample (30%) at the concentration of 24ug/ml. Note that the cytotoxicity of BU1 is still very high and masks the antiviral effect. KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151
  • 9. 5. Conclusions As seen in the first experiment, the sample with the most marked antiviral activity was BU1. The second experiment validated and the survival of the MT4 cells can be observed with different concentrations of BU1, TN1 and AQ1 samples. Partial protection of the cells can be seen with the BU1 sample (30%) at the concentration of 20ug/ml. However, the sample did not protect all the cells, because there are still many toxic compounds within it. Purification on the plant in natura is necessary, for the execution and repetition of the tests. ____________________________ Luiz Francisco Pianowski President KYOLAB Laboratório Ltda www.kyolab.com.br CNPJ: 05.758.608/0001-01 Laboratory: Rua Lauro Vannucci, nº 1020 – Jd. Sta. Cândida Campinas – SP CEP: 13087-548 Fone: 55 (19) 4062-8090 / (11) 4063-8090 Ramal: 1100 Administrative department: Av. José da Rocha Bonfim, nº 214 – Edifício Londres - Cond. Praça Capital Campinas- SP CEP: 13080-650 Fone/FAX: 55 (19) 37091151