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Inhibition of HIV-Replication in vitro and ex vivo by the plant extract TIB (Tian Immunity booster)

Information sourceTian Shengxun2013-01-30


 

REPORT
 
Inhibition of HIV-Replication in vitro and ex vivoby the plant extract TIB (Tian Immunity booster)
 
14. 11. 2005
 
by
 
PD Dr. Josef Schneider
Department of Virology
Institute for Medical Microbiology and Hygiene
University of Freiburg
Germany
 
Address:
 
Department of Virology, Institute for Medical Microbiology and Hygiene
Hermann-Herder-Str. 11 D-79104, Freiburg, Germany
Tel. Nr. ++49 761 203 6588
Fax Nr. ++49 761 203 6608
Institutional web-site: http://www.ukl.uni-freiburg.de/microbio
 
 
1. Summary
 
Water-soluble extracts of white and red pills made of TIB have been examined in two HIV inhibition tests in cell culture. The tests depend on the expression of the viral tat-gene and the entry of the virus into its host cells. In a third assay blood serum from a TIB-treated healthy person was examined for inhibition of HIV replication. Inhibition of HIV was observed in both cell culture tests at an IC50 of 0,15-0,5 mg/ml extract. Signs of cell toxicity were observed above 3 mg/ml. Furthermore, the blood serum of a treated person showed two-fold HIV inhibition in cell culture at 5-fold dilution as compared to serum before treatment. Thus the TIB preparation inhibits an early step of virus replication in cell culture and induces an antiviral activity in the blood of a treated person.

 

2. Experimental design
.
2.1 Extraction of pills:
Pills were pulverized with pestle and mortar. Phosphate buffered saline (PBS) was added and grinding continued. The insoluble material was sedimented and the soluble extract was filtered sterile. Filtrate was kept at 4°C until use for a maximum of 1 day.
The concentration of extracted materials was determined by lyophilization.
 
2.2 Virus replication assay:
Modified HeLa cells (HeLa-Tat-CD4-CXCR4 )were used as indicator cells for virus replication. They contained CD4, the HIV co-receptor CXCR4, and a reporter plasmid for HIV-Tat-expression.
If these cells are infected by HIV, the transactivator, Tat is expressed, which, in turn, will activate the reporter gene ?-Gal. Cells, in which HIV replicates are stained blue with the ?-Gal substrate X-Gal. The number of blue cells in a culture corresponds to the concentration of virus used to infect the culture.
 
2.3 Cell fusion assay:
CL4-cells, which have the glycoprotein complex Gp120-gp41 of HIV at their surface are co-cultured with the HeLa cells described in the replication assay. CL4 cells bind tightly to the HeLa-cells by use of the viral glycoprotein-receptor interaction. Subsequently the membranes of the cells fuse forming giant cells with at least two nuclei. Thus is a model of virus-cell fusion, in which the fusion process can be observed with the light-microscope by generation of giant cells and staining.
 
2.4 Treatment of a healthy person with TIB
Oral treatment with TIB may lead either to an uptake of herbal HIV-inhibitors into the blood, or to the induction of an antiviral response in the blood. In either case serum should have antiviral activity in the virus replication assay. Therefore in the virus replication assay fetal calf serum of the cell culture medium was replaced by sera from the treated person before and after treatment as well as serum of an untreated human to examine if antiviral activity was contained.
 

 

3. Experiments:
 
3.1 Antiviral Activity of water-soluble components of TIB pills
Date: August 26 - September 10, 2003
 
3.1.2 Extraction of pills and measurement of extracted materials
Weight of pills: white pill: 675mg, red pill: 677mg
Pulverize pills as described in 2.1 for 3 min, add 5 ml of PBS, and suspend slurry
Sediment slurry in centrifuge, 4000xg, 5 min., harvest supernatant.
Filtrate through membrane filter 0,22μm pore size.
Lyophilize 0,5 ml of filtrate for 5 hours, as reference lyophilize 0,5ml of PBS.
 
Results:
Table1:
Weight of solubilized and lyophilized material
 
weight
weight corr.
conc.
% of total
PBS
5,2 mg
--
 
 
White pill
38,7mg
33,5mg
67,0mg/ml
49,6
Red pill
48,6mg
43,4mg
86,8mg/ml
64,1
 
These results show that 49,6% of the white pills and 64,1% weight by weight of the white pills can be solubilized in phosphate-buffered saline (PBS).
 
3.1.4 Inhibition experiment
Serial 3-fold dilutions were made from the extracts of white and red pills in Cell culture medium. Culturing of cells: HeLa: In DMEM-standard + G418 1mg/ml, + puromycine 1 μg/ml.
300μl of medium containing 25000 of HeLa-Tat-CD4-CXCR4 cells were put into each well of a 48-well-plate. 50μl of the diluted extracts were added starting with the second dilution of 22,3 mg/ml white and 28,9 mg/ml red pill extract respectively. One hour later 50μl of virus suspension was added. Each assay was done in triplicate. Parallel cultures without virus were done to observe cytotoxicity. As a positive control a dilution series of AZT was established.
4 days later cells were fixed in 0,5% paraformaldehyde, washed and stained with x-Gal for 3 hours following a standard procedure. HIV-infected cells stained blue, and formed syncytia. Blue cells were counted with the microscope and the EC50 values for RES, WES and AZT were determined.
Concentrations of the inhibitors and the original data of the blue cell counts are provided in the following tables.
Table 2:
Final Concentrations of pill extracts in mg/ml and AZT in nanomolar as used in the assay:
 
C1
C2
C3
C4
C5
white extr.
2,8
0,93
0,31
0,103
0,034
red extr.
3,62
1,2
0,4
0,13
0,045
AZT (nm)
100
10
3
1
0,3
 
 
 
 
 
 
 
.Results:
 
Table 3:
Number of blue cells counted at end of assay
red1
red2
red3
whit1
whit2
whit3
AZT1
AZT2
red-m
whit-m
AZ-m
0
0
0
8
2
4
4
0
0
5
2
0
1
0
15
21
16
32
26
0
17
29
1
7
16
55
66
63
78
54
8
61
66
58
51
33
93
94
93
89
109
47
93
99
82
91
106
106
110
98
121
121
93
110
121
 
The mean value of blue cells in a triplicate culture without inhibitor was 101 per well.
Data of this experiment are summarized in Fig. 1 in which mean numbers of blue cells (table 3, last three columns ) were plottet against the final concentrations of the inhibitors.
 
One can see from Figure 1 that extracts of red and white pills can reduce the replication of the virus to zero or almost zero. The concentrations which lead to a reduction to 50% of the untreated value are 0,13mg/ml for the red pill extract and 0,42mg/ml for the white pill extract.
The parallel experiment with AZT shows that this assay measures inhibition with the best sensitivity reported for other tests , which measure viral antigen or viral nucleic acids, because 3-70 nm is the range of reported IC50-values for AZT.
 
3.2 Toxicity test with HeLa-Tat-CD4-CXCR4 cells
 
Toxicity of red and white pill extract was estimated within the experiment for HIV inhibition by omitting the infecting virus and observing cell morphology at day 4 after addition of the extracts. The cultures were set up in duplicates.
 
Results:
 
Table 4:
Dilutions of the inhibitors in mg/ml, (+) detection of cell damage and lack of growth
 
 
C1
C2
C3
C4
C5
C6
white extr.
5,6
2,8
0,93
0,31
0,103
0,034
dead cells white
+
-
-
-
-
-
red extr.
7,2
3,6
1,2
0,4
0,13
0,045
dead cells red
+
+/-
-
-
-
-
 
The data show that below about 3 mg/ml cell morphology and cell numbers did not change.
Because HeLa cells can tolerate about 3 mg/ml of both white and red pill extract the inhibition of virus replication at 0,13 and 0,42 mg/ml of the extracts should not be due to unspecific toxic effects.
 
 
 
3.3 Inhibition of HIV-envelope-protein induced cell fusion:
Date: November 16. 2003
.
Set-up of the assay:
CL4 cells were kept in DMEM-standard medium. For HeLa-Tat-CD4-CXCR4 cells G418 (1mg/ml) and puromycine (1 μg/ml) were added to this medium.
CL4-cells were trypsinized from a small t-bottle (5 min. in trypsine), suspended in 3 ml DMEM standard Med. The suspension was diluted to a density of 16.500 cells / ml in medium and 0,6 ml were added to each well of a 48-well microtiter plate. The plate was kept for 2-3 hrs. in the cell incubator.
By the same procedure a suspension of the modified HeLa cells was prepared.
0,3 ml medium were sucked off from the top of the settled CL4 cells without removal of the cells. , replaced by 300 μl of HeLa cell suspension (33.000 cells /ml), and 200μl of the respective inhibitor dilution in DMEM were immediately added.
Cultures were kept for 16-18 hours in the cell incubator to allow for syncytium formation.
Thereafter, cells were stained with hemocolor (Merck) following the manufacturers recommendations. Syncytia with more than 4 cell nuclei per cell were counted and pictures were taken of representative areas.
 
Results:
 
Table 4
Inhibition of Syncytium formation with increasing inhibitor concentrations
Concentrations of red and white pill extracts in mg/ml, concentration of
HS-1500 im μg/ml
 
Inhib-Conc
μg/ml
Syncyt
red
Syncyt
white
Syncyt
r + w
HS-1500
W/O
inhibitor
75
14,5
7,5
11
0
125
1,9
35
33
42
9
77
0,9
68
42,5
51
49
108
0,47
62
72,5
74
94
90
 
Columns 2 - 6 contain counted syncytia in each well of an 48 well microtiter plate
red = red pill extract, white = white pill extract, HS-1500 = other HIV-Inhibitor with
known syncytium inhibiting capacity.
W/O inhibitor = negative control: quadruplicate, average; 100 syncytia /well
 
The data of this table are graphically represented in Fig. 2.
Examples of syncytium containing cultures are presented in Figs 3a and 3b.
 
Comment to Fig. 2
 
Obviously dose response curves are obtained for the inhibition of the white and red pill extracts. However, the effective concentration of these plant products is about 1000-times higher than that of the synthetic humic acid homologue HS-1500.
In the assays ?Syncyt r+w a mixture of both extracts with half the concentration of each compound was used. Therefore the inhibitors have only additive effects. Because syncytium formation mimics virus cell entry and the efficacy of Syncytium inhibition is roughly the same as that of infection inhibition, one can assume that the compounds work mainly by preventing virus to cell binding or virus entry.
 
 
3. 4 Inhibition of HIV-1BaL replication with human Sera from a healthy individual obtained before and after TIB treatment
 
Date: 8. 1. 2004
 
Setup of the experiment:
A healthy person was treated with a high dose (30 pills of each daily) TIB red and white pills. At the day before onset of the treatment and the day thereafter blood was taken for serum preparation. The test was performed with sera stored at -80°C.
10.000 HeLa CD4- CXCR4- CCR5 cells were seeded with 300μl medium into each well of 48-well microtiter plates and incubated for 18 hrs. 90μl of original serum or two-fold serial dilutions thereof were added to the cells. 30 min thereafter virus stock was added that produced about 100 blue cells per well without inhibitor as described in experiment No. 1
At the day three after setup large syncytia were visible even without staining. Thus cultures were stained with X-Gal as described and blue cells were counted.
 
Results
Table 5: Number of blue cells in individual triplicate cultures
 
Ser.
Konc.
Ser. before Treatm.
Ser. after Treatm.
Ser. untreated control pers.
Fetal calf serum
20%
76
154
90
63
54
73
88
78
99
120
132
146
10%
138
62
81
65
52
74
88
69
84
139
148
132
5%
104
100
113
81
94
78
83
84
94
157
148
137
2,5%
121
144
116
133
129
139
109
139
150
149
153
142
1,25%
153
141
138
143
144
157
132
168
139
157
139
159
0%
133
149
145
185
168
144
162
158
146
170
152
146
 
The average blue cells in tests without human serum is 154,8 blue cells
The mean values of table 4 are graphically represented in Fig 4.
 
The figure 4 clearly shows that cultures with the serum of the treated person (Tian (+)) allows less virus replication than the serum of the same person before treatment (Tian (-)). Serum from another person has intermediate values. Furthermore clear dose response curves are obtained with all human sera indicating a systematic process that is most probably not due to statistical variation. In contrast increase of the fetal calf serum concentration has only little effect on the virus replication. Thus one can conclude that individual human sera may have different inhibiting effects on the replication of HIV-1, which is most probably enforced by treatment with TIB. These interesting data should be further corroborated with studies applying sera of other treated individuals.
 

 

 
4. Figure legends
 
Legend to Fig. 1 Inhibition of infection
Graphic presentation of the data in Table 3. Mean values of triplicate assays were plottet against the concentrations of the respective inhibitors from red pills, white pills and Azidothymidine (AZT) as comparison. Blue cells corrspond to virus replicating cells in individual cultures. Without inhibitor 100 - 120 blue cells are found in the cultures.
 
Legend to Fig. 2 Fusion inhibition with red / white CHM
CL4 cells and HeLa-CD4-CXCR4-cells were cocultured as described. Without inhibitor about 100 syncytia were counted after 16 hours of cultivation. The curves show the decrease of syncytia formed with increasing concentration of the indicated compounds.
 
Legend to Fig. 3: Fusion experiment, same experiment as Fig. 2
For comparison the effects of the known fusion inhibitors HS-1500 (synthetic humic acid and soluble CD4 (Fusion protein between CD4 and IgG-gamma chain) are also demonstrated.
Numbers of inserts:
1, HeLa -CD4-CXCR4-CCR5 cells alone
2, CL4 cells alone
3, Co-culture without inhibitor
4, Co-cult. with 18 μg/ml soluble CD4-H-gamma-1
5, Co-cult with 4.2 μg/ml humate HS-1500
6, Co-cult with 2.1 μg/ml humate HS-1500
7, 8, 9, Co-cultures with 3; 1.5; and 0.75 mg/ml white pill extract respectively
10, 11, 12, Co-cultures with 3.4; 1.7; and 0.84 mg/ml red pill extract respectively
 
Legend to Fig. 4: Inhibition of HIV by sera +/- TIB-treatment
HeLa -Tat-CD4-CXCR4-CCR5 cells were infected with HIV after pre-treatment with serial dilutions of the following sera (identified by symbols). Five days thereafter cells were fixed and stained with X-Gal to monitor HIV-replication.
 
Tian (-): Person before TIB-treatment (squares)
Tian (+): Person after TIB-treatment (diamonds)
JS: untreated control person (circles)
FKS: Fetal calf serum (triangles)