Protective effects of Cambodian medicinal plants on tert­butyl hydroperoxide­induced hepatotoxicity via Nrf2­mediated heme oxygenase­1.

Liver diseases are considered to be primary contributors to morbidity and mortality rates in humans. Oxidative stress is critical in liver injury, and oxidant­induced liver injury may be caused by toxins, including tert­butyl hydroperoxide (t­BHP). The present study investigated the hepatoprotective activities of 64 crude ethanol extracts of Cambodian medicinal plants against t­BHP­induced cytotoxicity in human liver­derived HepG2 cells, and assessed their cytoprotective mechanism pertaining to the expression of heme oxygenase (HO)­1 and nuclear factor E2­related factor 2 (Nrf2). Protective effects in HepG2 cells were determined by MTT assay. Protein expression levels of HO­1 and Nrf2 were determined by western blotting and mRNA expression levels were determined by reverse transcription­quantitative polymerase chain reaction. Of the 64 extracts, 19 extracts exhibited high hepatoprotective activities: Ampelocissus martini, Bauhinia bracteata, Bombax ceiba, Borassus flabellifer, Cardiospermum halicacabum, Cayratia trifolia, Cinnamomum caryophyllus, Cyperus rotundus, Dasymaschalon lomentaceum, Ficus benjamina, Mangifera duperreana, Morinda citrifolia, Pandanus humilis, Peliosanthes weberi, Phyllanthus emblica, Quisqualis indica, Smilax glabra, Tinospora crispa and Willughbeia cochinchinensis, with half maximal effective concentrations ranging between 59.23 and 157.80 µg/ml. Further investigations revealed that, of these 19 extracts, HO­1 and Nrf2 were expressed in P. weberi and T. crispa expressed in a dose­dependent manner. In addition, the activities of reactive oxygen species were suppressed following treatment of these two extracts in t­BHP­induced HepG2 cells. These results indicated that, of the 64 Cambodian plants, P. weberi and T. crispa exhibited hepatoprotective effects on t­BHP­induced cytotoxicity in HepG2 cells, possibly by the induction of Nrf2­mediated expression of HO­1. Taken together, these results suggested that T. crispa or P. weberi may offer potential for therapeutic applications in liver disease characterized by oxidative stress.

Simultaneous HPLC determination of three bioactive alkaloids in the Asian medicinal plant Stephania rotunda.

A reliable high-performance liquid chromatography (HPLC) method coupled with photodiode array detection has been developed and validated for the determination of three major alkaloids: cepharanthine, tetrahydropalmatine and xylopinine in Stephania rotunda Lour. (Menispermaceae) collected in Cambodia. The chromatographic separation was carried out on a Symmetry C8 column (250 mm x 4.6 mm, 5 microm, Waters), with an isocratic solvent system of 25 mM potassium phosphate buffer (pH 3.5) - acetonitrile. UV detection was performed at 282 nm. Good linear behavior over the investigated concentration ranges was observed with values of r2 > 0.9964 for all the analytes. The method was reproducible with intra- and inter-day variations of less than 3.91%. The mean recoveries of the analytes ranged from 95.7 to 104.6%. The proposed method was linear, accurate, precise and specific. The validated method was successfully applied to quantify the three alkaloids in various parts of Stephania rotunda and in tubers collected from different Cambodian regions. The results indicated that the developed HPLC method could be used for the quality control of S. rotunda.