Cell experiment:

HepG2 cells are treated with different concentrations of hispidulin (50, 100, 200 μM) for 24, 48 and 72 h. Following treatment, cells are further incubated with MTT reagents at 37℃ for 4 h before DMSO is added, to dissolve farmazan crystals, and absorbance is measured at 570 nm in a microplate reader[2].

Animal experiment:

Mice: Tumor are established in mice. Mice are treated with DMSO or Hispidulin at a dosage of 10, 20 or 40 mg/kg/day for 35 days. The body weight of tumour-bearing mice is recorded every week and tumour volume is calculated [2].

Hispidulin is a natural flavone with a broad spectrum of biological activities. Hispidulin is a Pim-1 inhibitor with an IC50 of 2.71 μM.

Hispidulin induces cell death in a dose- and time-dependent manner in HepG2 cells. Hispidulin induces apoptosis through mitochondrial dysfunction, which is characterized by decreased Bcl-2/Bax ratio, disrupted mitochondrial membrane potential and increased release of cytochrome C and activated capase-3[2].

Hispidulin shows significant inhibitory effect on mice tumor size[2]. Hispidulin treatment effectively prevents ovariectomy-induced body weight loss and attenuates ovariectomy-induced bone loss. Hispidulin treatment also decreases trabecular spacing in ovariectomy mice[3]. Intraperitoneally administering hispidulin(10 or 50mg/ kg) to rats 30 min before intraperitoneally injecting kainic acid (15mg/kg) increases seizure latency and decreases seizure score. In addition, hispidulin substantially attenuates kainic acid-induced hippocampal neuronal cell death, and this protective effect is accompanied by the suppression of microglial activation and the production of proinflammatory cytokines such as interleukin-1β, interleukin-6, and tumor necrosis factor-α in the hippocampus[4].

References:
[1]. Chao SW, et al. Total Synthesis of Hispidulin and the Structural Basis for Its Inhibition of Proto-oncogene KinasePim-1. J Nat Prod. 2015 Aug 28;78(8):1969-76.
[2]. Gao H, et al. Hispidulin induces apoptosis through mitochondrial dysfunction and inhibition of P13k/Akt signalling pathway in HepG2 cancer cells. Cell Biochem Biophys. 2014 May;69(1):27-34.
[3]. Zhou R, et al. Hispidulin exerts anti-osteoporotic activity in ovariectomized mice via activating AMPK signaling pathway. Cell Biochem Biophys. 2014 Jun;69(2):311-7.
[4]. Lin TY, et al. Protective effect of hispidulin on kainic acid-induced seizures and neurotoxicity in rats. Eur J Pharmacol. 2015 May 15;755:6-15.