Study On The Separation And Identification And Cytoxicity Of Amides From Black Pepper

Black pepper (Piper nigrum L.) has been paid wide attention in recent years since its various chemical components and pharmacological properties, including antimicrobial activity, anti-inflammatory activity, anticancer activity, hepatoprotective activity, bioavailability-enhancing, etc. Previous studies showed that the major components in Piper were amides, among which piperine was the main bioactive compound. The separation and purification of amides from plant materials are always complicated and usually require multiple chromatographic steps. To obtain the amides with high purity are very difficult. Therefore, It is very significant to establish a good method to separate and purify the amides from black pepper.In this study, high-speed countercurrent chromatography (HSCCC), Sephadex LH-20and preparative HPLC were successfully used for the large-scale separation and purification of the amides from black pepper. The details of the process are as follows:First, the pepper powders were extracted with85%ethanol. Acetic acid was added to make the extract acidified, and impurities were removed by chloroform. The remaining extract was partitioned with chloroform after suspended in the alkalified solution by NH4OH. The chloroform solution was concentrated under reduced pressure at50℃, which yielded105g of crude extract after dried in vacuum.Second, the chloroform extract was separated by HSCCC-D1200using a solvent system composed of petroleum ether-ethyl acetate-methanol-water (1:3:1.5:l,v/v) resulting seven sub-fraction FrA-FrG. FrE was identified as a pure compound with a purity of99.26%. FrD and FrF were further purified by preparative HPLC to yield compounds D and F. Other fractions were separated and purified by Sephadex LH-20-preparative HPLC to afford10compounds A1, A2, B1, B2, B3, B4, C1, C2, C3and G1. The structures of all the compounds were identified by MS,'H, and13C-NMR spectral data. The13compounds are Retrofractamide-A(A1), N-isobutyl-(2E,4E)-decadienamide(A2), Retrofractamide-C(B1), piperoleine A(B2),(2E,6E)-7-(1,3-benzodioxol-5-y1)-1-(piperidin-1-yl)hepta-2,6-dien-1-one (B3), Retrofractamide-B(B4), piperoleine B(C1), pipernonaline(C2), pipercyclobutanamides A(C3), Dehydropipernonaline(D), Piperine(E),(2E,4E,14Z)-N-Isobutyleicosa-2,4,14-trienamide (F), N-isobutyl-(2E,4E,12Z)-octadecatrienamide (G).Cytoxicity of compounds A1, Bl, B4, C2, D and E against lung cancer801D cells and murine macrophage J744.1cells were evaluated by MTT method. All compounds had weak cytotxicity against normal J744.1cells with IC50more than100μg/ml, and exhibited strong inhibition activity against801D cells with IC50between5-42μg/ml. Compounds A1, B4and C2with isobutyl group had stronger activity than compounds B1, D and E with piperidine ring, which suggested that isobutyl group may active group against the cytotoxic activity.