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Rhinacanthus nasutus (L.) Kurz has been used as a traditional medicine for treatment of various cancers in Thailand. Rhinacanthins (RNs), the main bioactive naphthoquinones in R. nasutus Kurz., possesses antiproliferation on many cancer cells and immunomodulatory activities. RNs are alcohol-soluble and not suitable for an oral intake. This study aims to encapsulate RNs into nanocapsules and examine the antiproliferation and immunomodulatory activities of the RNs-encapsulated nanocapsules. In order to understand the stability of RNs-encapsulated nanocapsules at gastric (pH 3.5), intestinal (pH 6.8 and 7.4) and storage (pH 5.5) pHs, the analyses of particle size by using dynamic light scattering analysis at 25℃exhibited that the nanocapsules had average particle sizes of 10.5 ± 0.11 nm at pH 5.5, which was not significantly different from those at pH 3.5, 6.8 and 7.4. In addition, the RNs-encapsulated nanocapsules were stable at both pH 3.5 and 5.5 for up to 4 hr. In presence of bile acid, the RNs-encapsulated nanocapsules was ruptured and flocculated at pH6.8 and 7.4 after holding for 1 and 0.5 hr, respectively, indicating that the RNs-encapsulated nanocapsules were capable to perform a pH-controlled release of RNs in an intestinal fluid.
When incubated with nanocapsules (with no RNs) for 24 hr, Caco-2 cells in long-time culture had a significantly higher viability than those in short-time culture. This is primarily because the differentiation of Caco-2 cells to a polarized monolayer is more complete with a long time in culture for which increases cell adaptability to nanocapsules. The viability of Caco-2 cells with 1 or 7 days in culture was higher for those incubated with polysaccharides-encapsulated nanocapsules (PNPs) than those with nanocapsules, indicating that the cytotoxicity decreased for nanocapsules with an additional coating of polysaccharides isolated from Lycium barbarum. RNs concentration even at a low level (1 ppm) had a proliferation effect on the rat intestinal epithelial cell line (IEC-6 cells) and maintained 80% cell viability at 100 ppm, implying that RNs had low cytotoxicity on IEC-6 cells. However, RNs imposed a strong inhibitory effect on both Caco-2 and human prostate cancer cells (PC-3 cells) with 65 and 39% reduction at 50 ppm. Similarly, PNPs at a 25-ppm level increased the IEC-6 cell growth to 107% and inhibited the growth of Caco-2 and PC-3 cells by 8 and 53%, respectively. When incubated with Polysaccharides-nanoencapsulated RNs (RNs-PNPs) at 10 and 25 ppm for 24 h, IEC-6 cells increased to 125 and 138%, Caco-2 cells reduced to 54 and 33% and PC-3 cells to 79 and 40%, respectively. If the incubation extended to 24, 48 and 72 h, the half maximum inhibitory concentration (IC50) was, respectively, found to be: 100, 75, 11.1 ppm for RNs, 26.7, 16, 15 ppm for PNPs and 21, 10.6, 9.8 ppm for RNs-PNPs. Lipopolysaccharides were commonly used to induce macrophage (RAW 264.7) to secret inflammatory cytokines of NO, IL-6 and TNF-α. Immunomodulation studies showed that inhibitory effects on NO, IL-6 and TNF-α production were, respectively, 2.3~44.4%, 14.8~36%, 67.4~88% for RNs and 60.3~83.2%, 24.8~36%, 19.3~85.5% for RNs-PNPs, manifesting that RNs and RNs-PNPs possessed anti-inflammatory and immunomodulatory activities. Results of flow cytometry indicated that fluorescence-labeled the cores of nanocapsules could transport into Caco-2 cells within 0.5 hr with above 10 of fluorescence average and the penetration rates of the core into cells increased as the incubation time and concentration of nanocapsules increased. Increase of penetration of labeled core in cells can well explain RNs-PNPs has a stronger inhibitory effect on both Caco-2 and PC-3 cancer cells than RNs.
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