The Hymenocallis littoralis, an ornamental and medicinal plant, had been traditionally used for wound healing. In the present study, an analytical method using HPLC with ultraviolet detection was developed for the quantification of lycorine in the extracts of different parts of wild plant and tissue culture samples of H. littoralis. The separation was achieved using a reversed-phase column. The method was found to be accurate, repeatable, and sensitive for the quantification of minute amount of lycorine present in the samples. The highest lycorine content was found in the bulb extract (2.54 ± 0.02 μg/mg) whereas the least was in the root extract (0.71 ± 0.02 μg/mg) of the wild plants. Few callus culture samples had high content of lycorine, comparable to that of wild plants. The results showed that plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 4.5 μM (2.58 ± 0.38 μg/mg) or a combination of 2,4-D at 9.00 μM with 4.5 μM of 6-benzylaminopurine (BAP), were the optimum concentrations for the production of high lycorine (2.45 ± 0.15 μg/mg) content in callus culture. The present analytical method could be of value for routine quantification of lycorine in the tissue culture production and standardization of the raw material or extracts of H. littoralis.
Crinum asiaticum Linne var. japonicum has long been used as a rheumatic remedy, as an anti-pyretic and as an anti-ulcer treatment, and for the alleviation of local pain and fever in Korea and Malaysia. In order to investigate the possibility of Crinum asiaticum Linne var. japonicum extract as a cosmetic ingredient, we measured its anti-inflammatory effect by its inhibition of iNOS (inducible nitric oxide synthase) and the release of PGE2, IL-6, and IL-8. We also measured its anti-allergic effect by its inhibition of beta-hexosamidase release. An HPLC experiment after extraction with 95% EtOH at pH 3.5 showed that Crinum asiaticum Linne var. japonicum was mainly composed of lycorine (up to 1%), a well-known immunosuppressor. The content of lycorine varied, depending on the type of plant tissue analyzed and the extraction method. In an anti-inflammatory assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)-activated mouse macrophage RAW 264.7 cells, the ethanol extract of Crinum asiaticum showed an inhibitory activity of NO production in a dose-dependent manner (IC50 = 58.5 microg/ml). Additional study by RT-PCR demonstrated that the extract of Crinum asiaticum significantly suppressed the expression of the iNOS gene. Moreover, the extract of Crinum asiaticum did not show any cytotoxicity, but did show a cell proliferation effect against LPS (a 10 approximately 60% increase in cell viability). In an assay to determine inhibition of the H2O2-activated release of PGE2, IL-6, and IL-8 in human normal fibroblast cell lines, the release of PGE2 and IL-6 was almost completely inhibited above concentrations of 0.05% and 1%, respectively. Moreover, the release of IL-8 was completely inhibited over the entire range of concentration (>0.0025%). In order to investigate the skin-sensitizing potentials of the extract of Crinum asiaticum, a human clinical test was performed after repeated epicutaneous 48-h applications under an occlusive patch (RIPT). The repeated and single cutaneous applications of Crinum asiaticum Linne var. japonicum extract under the occlusive patch did not provoke any cumulative irritation and sensitization reactions. The result showed that the extract of Crinum asiaticum Linne var. japonicum has a sufficient anti-inflammatory effect. Therefore, Crinum asiaticum Linne var. japonicum extract may be useful for development as an ingredient in cosmetic products.