Regarding the chemical structure of HBOA, it contains hydroxyl and imine bases that may act as either hydrogen bond donors or acceptors and play a role in the action of the drug and in the process of receptor binding. Calış et al
observed that the substitution of the benzene ring of benzoxazolinone derivatives by an acyl moiety resulted in enhanced anti-inflammatory and analgesic activities (20
). Therefore, we hypothesize that 3-substituted or 3,4-disubstituted derivatives of HBOA may possess a greater protective effect on the liver than HBOA.
Products were carbonized by concentrated sulfuric acid. In order to avoid carbonization and maintain the concentration of sulfuric acid, we added distilled water or filtrate when distilling. We obtained 2-nitroresorcinol by drip type water vapor distillation in order to avoid carbonising the product. HBOA was obtained from 2-nitroresorcinol by a ‘one-pot’ reduction reaction and subsequent cyclization with urea. In this reaction, hexahydrated ferric chloride-activated carbon was used as a catalytic agent and hydrazine hydrate was used as a reducing agent. We have previously compared the applicability of reduction with hydrazine hydrate with that of stannous chloride dissolved in hydrochloric acid; reduction by hydrazine hydrate was observed to be simpler and more convenient (21
). However, hydrazine hydrate must be evaporated to dryness to prevent it from reacting with urea. AcO-BOA and TC-3 were subsequently obtained by the reaction of HBOA with acetyl chloride, an acylating agent, in a basic triethylamine solution.
HPLC was used to determine the purity of the compounds. The method provides excellent separation of target compounds from unknown impurities with a resolution of >1.5 and a tailing factor range of 0.95–1.5. We observed that AcO-BOA and TC-3 are readily hydrolyzed in aqueous solution. Therefore, we employed a solution of CMC as required in order to avoid hydrolysis.
-induced liver injury is dependent upon reductive dehalogenation catalyzed by cytochrome p450 in the endoplasmic reticulum of hepatic cells leading to the generation of an unstable complex trichloromethyl radical (•
). The superoxide anion O2−
and the hydroxyl radical (•
OH) are reactive oxygen species (ROS) produced in mitochondria (4
). Lipid peroxidation is an important consequence of the metabolism of CCl4
). These oxygen radicals contribute to the process of lipid peroxidation. However, cells have various mechanisms for protecting themselves from the toxic effects of ROS, including free radical scavengers and chain reaction terminators such as SOD, CAT, GSH and Gpx systems (23
). Liver injury via the ROS pathway causes increases in the levels of ALT, AST, MDA and LDH, the syntheses of which are increased due to the extensive damage of the liver cells (4
The current study demonstrates that HBOA and its derivatives exhibit protective effects against acute liver injury, confirmed by the serum ALT and AST levels and H&E staining. Our investigation revealed that HBOA and its derivatives exhibit potent hepatoprotective effects against CCl4-induced liver damage in mice. This may be the result of increasing the activity of the antioxidant-defense system and the inhibition of lipid peroxidation.
Many hepatoprotective drugs have been of interest due to their antioxidant activity (25
). In the current study, the induction of acute liver injury by CCl4
increased the levels of MDA in liver tissue. MDA is a product of lipid peroxidation; elevated levels of MDA may reflect the degree of lipid peroxidation injury in liver cells (2
). High doses of HBOA and its derivatives are able to reduce the levels of hepatic MDA. Compared with the CCl4
model group, treatment with HBOA increased the activity or level of SOD, CAT, Gpx and GSH, which scavenge free radicals and simultaneously reduce lipid peroxidation, thus alleviating the oxidative damage caused by CCl4
. The results suggest that HBOA protects against the damage caused by the oxidation of hepatic cellular membranes by free radical scavenging. Treatment of the mice with the two derivatives of HBOA attenuated the hepatic SOD and GSH depletion induced by the intraperitoneal administration of CCl4
. Therefore, we hypothesize that the protective actions of the two derivatives are related to their ability to scavenge free radicals.
TNF-α activates various intracellular pathways which regulate inflammation, cell death and proliferation. In the liver, TNF-α not only mediates hepatotoxicity but also contributes to the restoration of functional liver mass by promoting hepatocyte proliferation and liver regeneration (26
). TNF-α expression was observed to be lower in the group treated with HBOA than in the model group, which demonstrates that the hepatoprotective effect of HBOA involved an anti-inflammatory mechanism and inhibiting the activity of secreted TNF-α on Kupffer cells (KCs).
In conclusion, these preliminary studies indicate that HBOA and its two acyl derivatives elicit a protective effect on CCl4-induced liver injury in vivo. This study supports the traditional use of Acanthus ilicifolius as an herbal remedy for various liver diseases. However, further studies are required in order to evaluate the mechanism of action these compounds.