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. 2014 Jun 27:14:205.
doi: 10.1186/1472-6882-14-205.

Immunomodulatory effect of an isolated fraction from Tinospora crispa on intracellular expression of INF-γ, IL-6 and IL-8

Walaa Najm Abood, Iman Fahmi, Mahmood Ameen Abdulla  1 , Salmah Ismail
Affiliations

Immunomodulatory effect of an isolated fraction from Tinospora crispa on intracellular expression of INF-γ, IL-6 and IL-8

Walaa Najm Abood et al. BMC Complement Altern Med. .

Abstract

Background: Immunomodulators are substances that modify immune system response to a threat. Immunomodulators modulate and potentiate the immune system, keeping it highly prepared for any threat. The immunomodulatory effect of the traditional medicine Tinospora crispa is investigated in this work.

Methods: T. crispa ethanol extract was fractionated by using different solvents. The ethanol extract and effective isolated fraction were used to investigate the potential immunomodulatory effect of different T. crispa doses ranging from 25 μg/mL to 1000 μg/mL on RAW 246.7 cells by detecting intracellular INF-γ, IL-6, and IL-8 expressions. The antioxidant activity of T. crispa was evaluated through FRAP and DPPH. The total phenolic and total flavonoid contents were also quantified.

Results: Results show that T. crispa extract has higher antioxidant potential than ascorbic acid. The FRAP value of T. crispa extract is 11011.11 ± 1145.42 μmol Fe(+2)/g, and its DPPH inhibition percentage is 55.79 ± 7.9, with 22 μg/mL IC50. The results also reveal that the total phenolic content of T. crispa extract is 213.16- ± 1.31 mg GAE/g dry stem weight, and the total flavonoid content is 62.07- ± 39.76 mg QE/g dry stem weight. T. crispa crude extract and its isolated fraction significantly stimulate RAW264.7 cell viability (P ≤ 0.05) and intracellular INF-γ, IL-6, and IL-8 expressions. The results of LC-MS show that four of the active compounds detected in the T. crispa isolated fraction are cordioside, quercetin, eicosenoic acid (paullinic acid), and boldine.

Conclusions: The results of this study obviously indicate that T. crispa has immunomodulatory effects through the stimulation of INF-γ, IL-6, and IL-8 expressions. LC-MS phytochemical analysis showed that the T. crispa fraction has cordioside, quercetin, eicosenoic acid (paullinic acid), and boldine, which may be responsible for the immunostimulator effect of T. crispa.

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Figures

Figure 1
Figure 1
Tinospora c rispa stem and leaves.
Figure 2
Figure 2
Cell viability Percentage of RAW264.7 cell treated groups of T. crispa compared to control (untreated group). Each value represents the mean percent ± S.D.*significantly different versus control group, P ≤ 0.05.
Figure 3
Figure 3
Cell viability Percentage of RAW264.7 cell treated groups of T. crispa (T. c) F1, F2, F3, F4 and F5 compared to control (untreated group). Each value represents the mean percent ± S.D.*significantly different versus control group, P ≤ 0.05.
Figure 4
Figure 4
Intracellular expression of INF-γ on RAW264.7 macrophage cell. Flow cytometry analysis was used to assess the intracellular expression of INF-γ. The figures show the expression percent of INF-γ on RAW264.7 cell stimulated with (A) 1 μg/ml LPS alone as a control. (B) 1 μg/ml LPS + T. crispa 100 μg/ml. (C) LPS1μg/ml + T. crispa F2 100 μg/ml. The number was represented the mean percent of the cell ± SD. *Significant P ≤ 0.05 versus control.
Figure 5
Figure 5
Intracellular expression of IL-6 on RAW264.7 macrophage cell. Flow cytometry analysis was used to assess the intracellular expression of IL-6. The figures show the expression percent of IL-6 on RAW264.7 cell stimulated with (A) 1 μg/ml LPS alone as a control. (B) 1 μg/ml LPS + T. crispa 100 μg/ml. (C) LPS1μg/ml + T. crispa F2 100 μg/ml. The number was represented the mean percent of the cell ± SD. *Significant P ≤ 0.05 versus control.
Figure 6
Figure 6
Intracellular expression of IL-8 on RAW264.7 macrophage cell. Flow cytometry analysis was used to assess the intracellular expression of IL-8. The figures show the expression percent of IL-8 on RAW264.7 cell stimulated with (A) 1 μg/ml LPS alone as a control. (B) 1 μg/ml LPS + T. crispa 100 μg/ml. (C) LPS1μg/ml + T. crispa F2 100 μg/ml. The number was represented the mean percent of the cell ± SD. *Significant P ≤ 0.05 versus control.
Figure 7
Figure 7
Mass spectrum (TOF MS ES+) and chemical structure of cordioside (peak No. 1) identified in T. crispa F2.
Figure 8
Figure 8
Mass spectrum (TOF MS ES+) and chemical structure of quercetin (peak No. 2) identified in T. crispa F2.
Figure 9
Figure 9
Mass spectrum (TOF MS ES+) and chemical structure of Eicosenoic acid (paullinic acid) (peak No. 3) identified in T. crispa F2.
Figure 10
Figure 10
Mass spectrum (TOF MS ES+) and chemical structure of boldine (peak No. 4) identified in T. crispa F2.
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