Background: Benjakul, a Thai traditional herbal preparation, comprises five plants: Piper chaba, Piper sarmentosum, Piper interruptum, Plumbago indica, and Zingiber officinale. It has widely been used to treat cancer patients in folk medicine in Thailand. Benjakul extract, and its isolated compounds should be investigated for cytotoxic activity and analysis isolated compounds from chemical fingerprinting.
Objective: To study cytotoxicity of Benjakul extract and its isolated pure compounds against human small cell lung cancer cell line (NCI-H1688) and in normal human lung fibroblast cell line (MRC-5) and analysis the content of isolated compounds for quality control of Benjakul extract.
Material and Method: Bioassay-guided fractionation was used for isolated active compounds from ethanolic extract of
Benjakul. Cytotoxic activity was carried using the SRB assay. HPLC method was applied to analyze six isolated compound content from Benjakul extract.
Results: The ethanolic extract of Benjakul showed cytotoxicity against NCI-H1688 with IC50 value = 36.15+4.35 μg/ml. Hexane fraction as semi-separation by VLC showed the best cytotoxic activity (21.17+7.42 μg/ml). Six isolated compounds were identified as myristicin, plumbagin, methyl piperate, 6-shogaol, 6-gingerol and piperine. Plumbagin exhibited the highest cytotoxic activity and 6-shogaol was the second most effective cytotoxic constituent (IC50 values = 1.41+0.01 and 6.45+0.19 μg/ml, respectively). Piperine showed the highest content in both of HPLC analysis and column chromatography separation.
Conclusion: Benjakul extract exhibited cytotoxicity against NCI-H1688. Plumbagin and 6-shogaol are bioactive markers for cytotoxicity against this small cell lung cancer cell line. Chromatographic fingerprinting can be used to analyze six cytotoxic compounds isolated from the ethanolic extract of Benjakul.

Keywords: Benjakul, Cytotoxic activity, NCI-H1688, Chromatographic fingerprint