Document Type

Thesis - University Access Only

Award Date

2005

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry

Abstract

Cucurbit plant extracts have been used against various liver disorders in traditional medicine and their activity was thought to be due to the presence of cucurbitacin. Only a few liver protection studies were conducted on cucurbitacin in vivo. This is the first investigation to study cucurbitacin in vitro hepatoprotective activity on human hepatocyte-derived HepG2 and rat stellate cells-derived HSC-T6 cells. A number of cucurbitacin analogues were isolated from Cucurbita texana and Citru/lus Janatus. Semi-synthetic cucurbitacin were generated by alkylation, acetylation, and enzymatic hydrolysis. Cucurbitacin cytoprotection of HepG2 cells against CCl4- induced toxicity, and cucurbitacin anti-proliferative activity on HSC-T6 cells were studied. Cucurbitacin cytotoxicity was also measured to identify the non-toxic concentration for hepatoprotective activity screening. The ICso of cucurbitacin on HepG2 cells ranged between 4 μM and 390 μMand between 2 μMand 256 μMon HSC-T6 cells. Natural analogs showed an 11-to-192 fold higher toxicity on Hela cells than on HepG2 cells confirming the differential cytotoxicity of cucurbitacin. Our study indicated that the majority of cucurbitacin had cytoprotective activity on HepG2 cells and the most active ten compounds EC50 ranged between 2.4 μMand 45.3 μM. A 3-to-9 folds less concentration than their IC50 was necessary to achieve 50% cytoprotection on HepG2 cells. Cucurbitacin demonstrated side effects on cell morphology at their ECso concentration. In order to reduce toxicity and improve the margin between active and toxic dose, future studies should focus on other structural modifications. All cucurbitacin analogs demonstrated high and novel anti-proliferation effect on serum-activated HSC-T6, and their EC50 ranged between 0.02 and 4.12 μM. An 8-to-344 folds less concentration-than their IC50 was necessary to achieve 50% anti-proliferation on HSC-T6 cells. No morphological changes were observed for the majority of the analogs. On both cell lines, the alkylated and alkyl-acetaylated derivatives showed higher toxicity and lower or no activity. The rest of the cucurbitacin proved to have high potential as liver anti-fibrosis agents. Cucurbitacin demonstrated no antioxidant activity. Certainly, cucurbitacin protect against hepatocellular injury by a different pathway than the radical scavenging mechanism or anti-lipid peroxidation. Cucurbitacin anti-inflammatory activity may be involved in the liver protection. However, this activity is not apparent from the protection of the connective tissue against the destructive effect of hyaluronidase enzyme. The effect of lipophilicity on cytotoxicity was studied. We found that cucurbitacin lipophilicity correlated to a great extent with the cytotoxicity measured on HepG2 and HSC-T6 cell lines. They demonstrated a wide range of lipophilicity and logP ranged from 2.09 to 4.93. A chromatographic approach of measuring lipophilicity proved to be of high-throughput, highly precise, and fast. Acetonitrile organic phase yielded greater peak resolution, and the shorter column yielded higher accuracy. The correlation between lipophilicity and cytotoxicity demonstrated that increasing lipophilic character of derivatives resulted in an increase in cytotoxicity. An exception was the new C16-acetyl derivatives, which proved to be more lipophilic but less toxic than the original compounds. QSAR equations were established for the models involving cytotoxicity or hepatoprotective activity of cucurbitacin derivatives. Other physicochemical properties than lipophilicity were determined by ab initio calculations. Lipophilicity was found to be the major descriptor for models involving cytotoxicity and cytoprotection on HepG2 cells. Lipophilicity was also found to be the major descriptor in the model describing cytotoxicity on HSC-T6. Surface area was found to be the major descriptor for the model describing the anti-proliferation activity on HSC-T6 cells. Descriptors other than lipophilicity also correlated with bioactivity such as atomic net charges and surface area. The QSAR equations were found to be statistically significant and were further used to predict the bioactivity of novel cucurbitacin derivatives.

Library of Congress Subject Headings

Cucurbitaceae
Liver - Diseases - Treatment

Format

application/pdf

Number of Pages

201

Publisher

South Dakota State University

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