Zerumbone- (ZER-) loaded nanostructure lipid carrier (NLC) (ZER-NLC) prepared for its antileukemia effect in vitro was evaluated for its toxicological effects by observing changes in the liver, kidney, spleen, lung, heart, and brain tissues, serum biochemical parameters, total haemogram, and bone marrow stem cells. The acute toxicity study for ZER-NLC was conducted by orally treating BALB/c mice with a single dose with either water, olive oil, ZER, NLC, or ZER-NLC for 14 days. The animals were observed for clinical and behavioral abnormalities, toxicological symptoms, feed consumption, and gross appearance. The liver, kidney, heart, lung, spleen, and brain tissues were assessed histologically. Total haemogram was counted by hemocytometry and microhematocrit reader. Bone marrow examination in terms of cellular morphology was done by Wright staining with bone marrow smear. Furthermore, serum biochemical parameters were determined spectrophotometrically. Grossly all treated mice, their investigated tissues, serum biochemical parameters, total haemogram, and bone marrow were normal. At oral doses of 100 and 200 mg/kg ZER-NLC there was no sign of toxicity or mortality in BALB/c mice. This study suggests that the 50% lethal dose (LD50) of ZER-NLC is higher than 200 mg/kg, thus, safe by oral administration.
Zerumbone (ZER) isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl- β -cyclodextrin (HP β CD) to enhance ZER's solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HP β CD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HP β CD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HP β CD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.
The current study was designed to evaluate the in vitro cytotoxicity effect of a phenylbutenoid dimer, cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-3 (‴) ,4 (‴) -dimethoxystyryl]cyclohex-1-ene (ZC-B11) isolated from the rhizome of Zingiber cassumunar on various cancer cell line, and normal human blood mononuclear cells, and to further investigate the involvement of apoptosis-related proteins that leads, to the probable pathway in which apoptosis is triggered. Cytotoxicity test using MTT assay showed selective inhibition of ZC-B11 towards T-acute lymphoblastic leukemia cells, CEMss, with an IC50 value of 7.11 ± 0.240 μ g/mL, which did not reveal cytotoxic effects towards normal human blood mononuclear cells (IC50 > 50 μ g/mL). Morphology assessments demonstrated distinctive morphological changes corresponding to a typical apoptosis. ZC-B11 also arrested cell cycle progression at S phase and causes DNA fragmentation in CEMss cells. Decline of mitochondrial membrane potential was also determined qualitatively. In the apoptosis-related protein determination, ZC-B11 was found to significantly upregulate Bax, caspase 3/7, caspase 9, cytochrome c, and SMAC and downregulate Bcl-2, HSP70, and XIAP, but did not affect caspase 8, p53, and BID. These results demonstrated for the first time the apoptogenic property of ZC-B11 on CEMss cell line, leading to the programmed cell death via intrinsic mitochondrial pathway of apoptosis induction.