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Microbes & Immunity Biological activity of Amazonian plants

2.5. Cytokine quantification (cell counting/Trypan blue staining). An optimal

EHLC was treated with an optimal, low-toxicity dilution of 1:1024, corresponding to 0.34 µg/mL for
concentration of plant extract and infected with both botanical species, was utilized to treat the EHLC
SIVmac251 after 24 h. Supernatant samples were harvested in culture. Cells were counted at time intervals ranging
at 48, 144, and 240 h post-infection. The control groups from 48 to 288 h post-treatment with plant extracts.
included supernatant from uninfected cells and simian Regarding LM extract treatment, at the initial and final
immunodeficiency virus (SIV)-infected cells, SIV- time intervals of 48 and 288 h, treated cells had 1.5 times
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uninfected and plant extract-untreated cells, as well as more cells than in the control (1.5 × 10 treated cells
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SIV-uninfected and plant extract-treated cells. Plate wells vs. 1.0 × 10 untreated cells) and 2.3 times more cells
than in the control (1.6 × 10 treated cells vs. 0.7 × 10
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were sensitized overnight at 4°C with capture antibodies untreated cells), respectively, with statistically significant
for each IL-4, IL-6, IL-8, IL-10, and IFN-γ, diluted in differences (p=0.0133 and p=0.0122, respectively). At the
PBS according to the kit instructions (DuoSet ELISA 96-h time point, there was no significant difference in cell
Development System, R&D Systems, United States). density between treated and untreated cells (p=0.0842).
After 4 times washes with buffer, the blocking solution At intermediate intervals of 144, 192, and 240 h, LM
(PBS-BSA 1 – 0.05% NaN ) was added. Following a 1-h extract-treated cells exhibited reduced growth than
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incubation at room temperature, the wells were washed in the control, with statistically significant differences
again, and reference ILs and leukocyte cell culture (p=0.0069, p=0.0177, and p=0.0039, respectively). Cells
supernatant were added to each well in triplicate. Samples treated with OH extract had exceptional cell growth at 48
were incubated at room temperature for 2 h. Subsequently, and 96 h post-treatment compared to the control, with
diluted detection antibody solution was added to each increases of about 3.3 times and 1.4 times (p=0.0182 and
washed well. After 2-h incubation, streptavidin-peroxidase p=0.0087, respectively). At 192 h, the growth of treated
solution was introduced, followed by a 20 min incubation cells dropped from 4.6 × 10 to 2.8 × 10 cells/mL, while
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and subsequent washing. Chromogenic substrate solution untreated cells remained at 4.4 × 10 cells/mL (p=0.0087).
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(TMB) was then added, protected from light exposure, At the final time interval of 288 h, OH extract-treated
and incubated for 30 min. Sequentially, the reaction was cells continued growing, reaching 1.5 × 10 cells/mL,
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stopped with sulfuric acid solution, and absorbance was whereas the untreated cell growth declined to 0.5 ×
measured at 450 nm using a multiplate spectrophotometer 10 cells/mL (p=0.0033). Comparing the mitotic activity
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(Thermoplate, Italy), with results expressed in optical between LM and OH extract treatments, OH extract-
density. These results were further confirmed by treated cells generally showed higher cell density than in
immunoassays for the same cytokines by utilizing ELISA LM extract-treated cells. From 48 to 192 h, the mitotic
kits kindly donated by ImmunoTools GmbH. 27 activity difference between OH and LM extract-treated
2.6. Statistical analysis cells reduced from 2.4 times to 1.5 times in favor of OH
extract treatment (p=0.018, p=0.0074, and p=0.0170,
The obtained data were analyzed by utilizing GraphPad respectively). At the final intervals of 192 – 288 h, the
Prism software (version 10.4, US) and subjected to two- difference in cell proliferation between the treatments
way ANOVA, followed by Tukey’s multiple comparison was minimal (p=0.1001 and p=0.0125) (Figure 2).
tests, where appropriate.
EHLC protection against lentivirus infection
3. Results (SIVmac251) and/or viral protein synthesis, following
treatment with LM and OH extracts, was evaluated
The UHPLC-PDA-MS analysis revealed betulinic acid based on cell survival and cytopathic effects. LM extract
and niruriflavone as the most abundant compounds treatment had significant protective effects at 144 h and
in the leaf extract of LM. Additional compounds 192 h (p=0.0127 and p=0.0476, respectively), while OH
identified by comparison with the literature data included extract treatment reduced the viral cytopathic effect at 96 h
(-)-gallocatechin, (-)-epigallocatechin, and (-)-4’-O-methyl- (p=0.0144). However, at the final time points of 240 h and
epigallocatechin.
288 h, neither extract provided significant protection to
Previously, cytokine production was not detected in the the cells (Figure 3).
Hut-78 cell line supernatant after stimulation with plant Cytokine expression by cells after treated with
extracts and SIV infection. Therefore, EHLC was generated plant extracts and challenged with lentivirus infection
to carry out experiments to detect cytokine production. (SIVmac251), along with controls represented by
The optimal plant extract dilution was determined untreated and SIV-infected cells, showed the following
based on cytotoxic assay results and phenotypic analysis patterns. Regarding cells culture treated with OH extract

Volume 2 Issue 2 (2025) 67 doi: 10.36922/mi.8367