Potent anti-HIV activities and mechanisms of action of a pine cone extract from Pinus yunnanensis

Abstract

The anti-HIV activities of a pine cone extract (YNS-PY-F) from Pinus yunnanensis have been evaluated, and its mechanisms of action were also explored. The pine cone extract, YNS-PY-F, potently inhibited HIV-1(IIIB), HIV-1(RF), HIV-1(A17), HIV-1(AO18) and HIV-2(ROD) and induced cytopathic effect in C8166 cells with EC₅₀ values of 0.96 μg/mL, 1.53 μg/mL, 0.88 μg/mL, 7.20 μg/mL and 6.17 μg/mL, respectively. The quantification of a p24 production assay showed that YNS-PY-F significantly inhibited the acute replication of HIV-1(IIIB), HIV-1RF, HIV-1(A17) and HIV-1(AO18) in C8166 cells. An MTT assay showed that YNS-PY-F also significantly inhibited the HIV-1(IIIB) induced cytolysis in MT-4 cells with an EC₅₀ value of 2.22 μg/mL. The mechanism assays showed that YNS-PY-F had potent inhibitory effects on the fusion between infected cells and uninfected cells, and the activity of HIV-1 reverse transcriptase, with EC₅₀ values of 7.60 μg/mL and 4.60 μg/mL, respectively. Overall, these data suggest that the pine cone extract from Pinus yunnanensis has potent inhibitory activities against HIV-1(IIIB), HIV-1(RF), RT inhibitor-resistant strains HIV-1(A17) and HIV-1(AO18), and HIV-2(ROD), and its anti-HIV mechanisms include inhibition of HIV entry and inhibition of reverse transcriptase activity.

Figure 1

Cytotoxities and anti-HIV activities of YNS-PY-F. Cytotoxities on C8166 and MT-4 cells were measured by MTT assay (A); Inhibitory activities on HIV-1_IIIB, HIV-1_RF, HIV-1_A17, HIV-1_AO18 and HIV-2_ROD in C8166 cells were measured by syncytia reduction assay (B); Inhibitory activities on acute replication of HIV-1_IIIB, HIV-1_RF, HIV-1_A17 and HIV-1_AO18 in C8166 cells were measured by quantification of p24 antigen production (C); Inhibitory activity on HIV-1_IIIB induced cytolysis in MT-4 cells was measured by MTT assay (D). The data shown in the figure are a representative of at least two independent experiments.

Figure 2

Inhibitory effects of YNS-PY-F on cell-to-cell fusion and RT activity. Inhibitory activity on the fusion of uninfected C8166 cells and H9 cells chronically infected HIV-1_IIIB was measured by syncytia reduction assay (A); Inhibitory activity on recombinant HIV-1 reverse transcriptase was measured by ELISA assay (B). The data shown in the figure are a representative of at least two independent experiments.

Figure 3

Effect of YNS-PY-F on the nuclear import of HIV-1 integrase in HeLa cells. (A) Negative control: EGFP-IN mainly distributed in the nucleus of HeLa cells; (B) D77 at 100 μg/mL: EGFP-IN mainly distributed in the cytoplasm of HeLa cells; (C) YNS-PY-F at 200 μg/mL: EGFP-IN mainly distributed in the nucleus of HeLa cells. The scale bar was 25 μm.

Figure 4

Effect of YNS-PY-F on SDF-1α induced CXCR4 internalization. (A) Negative control: EGFP-CXCR4 mainly distributed in the cytoplasm of 293 T cells treated by SDF-1α; (B) AMD3100 at 10 μM: EGFP-CXCR4 still mainly distributed in the cell membrane of cells treated by SDF-1α and AMD3100; (C) YNS-PY-F at 200 μg/mL: EGFP-CXCR4 mainly distributed in the cytoplasm of 293 T cells treated by SDF-1α andYNS-PY-F. The scale bar was 25 μm.

Figure 5

The structure of lignin-carbohydrate complex [16]. Carbohydrate represents polysaccharide portion of lignin–carbohydrate complex.