project mutamba aids\ hiv

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