Kleeb Bua Daeng formula is a popular traditional remedy sold by the Chao Phya Abhaibhubejhr Hospital, Thailand. This formula contains Piper nigrum L., Nelumbo nucifera Gaertn. and Centella asiatica L. components. A reliable and accurate analytical method for the determination of five major active compounds in this formula was developed and validated. High performance liquid chromatography with a diode-array detector was used to quantify piperine, quercitin, kaempferol, asiaticoside and madecassoside in Kleeb Bua Daeng formula. The separation was carried out using a hypersil C18 column with detection at wavelengths 210, 280 and 370 nm. Gradient chromatographic conditions using a mixture of 0.05% phosphoric acid and acetonitrile allowed for complete resolution of the 5 active compounds. The validation outcomes for accuracy, precision, linearity, limit of detection, limit of quantitation and robustness demonstrated that this HPLC method was accurate and reliable for the simultaneous determination of active compounds in this formula. Hence, this developed and validated HPLC fingerprint method is appropriate for quality control of Kleeb Bua Daeng formula and could be applied to modified Kleeb Bua Daeng formulations.
Kleeb Bua Daeng formula is one of the popular remedies sold in Chao Phya Abhaibhubejhr Hospital, Thailand. This formula contains Piper nigrum L., Nelumbo nucifera Gaertn., and Centella asiatica L. as active components. Owing to getting the highest content of its phytochemical compounds, the conditions of solvent extraction for this formula were optimized. The type of solvent, number of extraction times, and ratio between the material and solvent were varied in this study using the Box–Behnken design. The important phytochemical constituents (total phenolics, flavonoids, carotenoids, and anthocyanins) were also determined. From the result of this study, it was found that the highest content of each total active compound was obtained from different conditions such as the optimal extraction condition of phenolic content was obtained using methanol as solvent, one time of extraction, and the ratio of powder and solvent was 1 : 6. Thus, the variation of solvent extraction condition could affect the phytochemical content. Further studies about the herbal formula involving the extraction process should concern the variation of extraction conditions to get the highest content of the active compound.
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