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PARTIAL PURIFICATION OF ANTICARCINOGENIC COMPONENT(S) OF YOGURT
Abstract
Previous studies in our laboratory have established that feeding yogurt to mice bearing Ehrlich ascites tumor cells (EATC) resulted in statistically significant (P < 0.05) reduction of tumor growth and proliferation, when compared to the control group fed only milk. The present series of investigations were therefore conducted to isolate and concentrate the active antitumor component(s) of yogurt. Initially, yogurt was dialyzed against deionized distilled water, yielding dialyzate, and retenate (residue) fractions. The fractions were lyophilized, and their antitumor activities determined by feeding to mice, previously inoculated with EATC. The proliferation of the tumor was monitored by both total cell count and deoxyribonucleic acid (DNA) content of ascitic fluid recovered from the peritoneal cavity. Feeding of the dialyzate fraction resulted in statistically significant (P < 0.05) inhibition than the retentate fraction on cell cound (24.3% and 5.3%, respectively), as well as on DNA content of ascitic fluid suspension (32.3% and 7.1%, respectively), when compared to the control group fed milk. A spectrophotometric scan of yogurt dialyzate in the ultraviolet range revealed two peaks, one at 280 nm and the other at 212 nm. A similar scan of milk dialyzate did not result in any discernible peaks. Another method of separating the antitumor compounds from yogurt was also investigated, using petroleum ether as the solvent. Extraction of yogurt in petroleum ether resulted in an aqueous ether-insoluble fraction. A single intravenous injection of 2 mg/mouse of lyophilized aqueous ether-insoluble fraction caused 25.1% to 32.3% reduction (significant P < 0.05) in tumor cell growth when compared to the control group injected with saline. In subsequent trials, yogurt dialyzate which was fed in the preceding studies was separated into acidic, basic, and neutral compounds by ion exchange chromatography. The antitumor activity of these fractions was determined using both in vitro and in vivo methods. The effect of a single intravenous injection of 2.5 mg per mouse of yogurt dialyzate and ion exchange fractions into mice bearing Ehrlich ascites tumor cells was determined. Enumeration of ascites tumor growth after 7 days revealed that the yogurt dialyzate and the acidic (anionic) fraction of dialyzate resulted in 38.5% and 38.7% reductions of cell numbers, respectively. The neutral fraction did not result in any inhibition, while the basic (cationic) fraction resulted in only 7.5% reduction. Two in vitro techniques, Agar Plate Diffusion (ADP) and Cell Culture (CC), were also employed to determine whether the fractions which exhibited activity in the mouse assay were active in vitro. In the ADP method, the ascites tumor cells were grown in "Miyamura" agar. Paper discs saturated with dialyzate, ion exchange fractions, and mercuric chloride (used as a stardard) were placed on the agar and incubated. The zone of inhibition produced by the failure of dead cells to reduce methylene blue dye was measured. Only mercuric chloride produced a measureable zone (15.7 mm), while yogurt dialyzate and ion exchange fractions failed to exhibit any measurable inhibition. Similarly, in the cell culture technique, mercuric chloride caused a drastic reduction (significant P < 0.05) in cell viability from 70.0% to 4.6% in 120 min. Yogurt dialyzate and ion exchange fractions did not produce any reduction in cell viability. The ability of dialyzate and ion exchange fractions to cause inhibition in the in vivo test (mouse assay) but not in the in vitro assays may be due to the elicitation of immunological response of the host by the antitumor fraction.
Subject Area
Food science
Recommended Citation
AYEBO, AMADU DERRICK, "PARTIAL PURIFICATION OF ANTICARCINOGENIC COMPONENT(S) OF YOGURT" (1980). ETD collection for University of Nebraska-Lincoln. AAI8021332.
https://digitalcommons.unl.edu/dissertations/AAI8021332