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Cytotoxicity and bioavailability studies on a decoction of Oldenlandia diffusa and its fractions separated by HPLC.

Summary

AIM OF THE
STUDY:
Oldenlandia diffusa is a traditional Chinese herbal remedy with known cytotoxic activity in vitro and in vivo. The aim of the study was to identify the most cytotoxic constituents of a water extract (a decoction is traditionally used as a treatment) by HPLC and activity-guided fractionation. The bioavailability of the decoction and certain fractions, and the mode of cell death induced by these mixtures, were also investigated. MATERIAL AND
METHODS:
A decoction of O.diffusa was prepared and separated by HPLC into eleven fractions (F1-11) for testing on the growth of HL60 leukaemia cells; two of the most active fractions were also tested on Caco-2 colon cancer cells. Cell viability was measured by trypan blue exclusion, DNA content (Cyquant NF assay) and neutral red uptake. Evidence of apoptosis was gained from cells stained with the nuclear dye DAPI, and detection of cleaved poly (ADP-ribose) polymerase (PARP) by Western blot.
RESULTS:
Fraction 9 was found to be the most active fraction, and, along with the decoction, induced apoptosis. Cells stained with DAPI showed a decrease in cell size and nuclear fragmentation characteristic of apoptosis. Detection of cleaved PARP further confirmed induction of apoptosis by O.diffusa decoction and fraction 9. The bioavailability of O.diffusa was investigated by production of post-absorption samples using Caco-2 intestinal epithelial monolayers. Addition of post-absorption samples (taken from the basolateral side after 3 hr incubation with the decoction on the apical side) inhibited the growth of HL60 cells, and suggested a degree of bioavailability. The constituents in fraction 9 were further separated by HPLC and eight major compounds were identified by LC-
MS:
two of these were ursolic acid (UA) and its enantiomer oleanolic acid (OA). Concentrations of UA and OA in the decoction were then calculated by reference to the area of the peaks of UA and OA found in F9. The post-absorption sample of F9 contained six of the eight constituents in the original pre-absorption fraction 9.
CONCLUSIONS:
Taken together, the results suggest that certain constituents, possibly including ursolic/oleanolic acid, may be bioavailable and at sufficient concentration to induce apoptosis in cancer cells in vitro through a mechanism including the cleavage of PARP.

Affiliation

School of Pharmacy & Chemistry, Kingston University, Penryhn Road, Kingston upon Thames, Surrey KT1 2EE.

Journal Details

Name: Journal of ethnopharmacology
ISSN: 1872-7573
Pages:

Links

MESH Definitions

Gliadin

Simple protein, one of the prolamines, derived from the gluten of wheat, rye, etc. May be separated into 4 discrete electrophoretic fractions. It is the toxic factor associated with CELIAC DISEASE.

Epidemiologic Studies

Studies designed to examine associations, commonly, hypothesized causal relations. They are usually concerned with identifying or measuring the effects of risk factors or exposures. The common types of analytic study are CASE-CONTROL STUDIES; COHORT STUDIES; and CROSS-SECTIONAL STUDIES.

Hedyotis

A plant genus of the family RUBIACEAE. Members contain anthraquinones and iridoids. H. diffusa is used in DRUGS, CHINESE HERBAL.

Heparin, Low-molecular-weight

Heparin fractions with a molecular weight usually between 4000 and 6000 kD. These low-molecular-weight fractions are effective antithrombotic agents. Their administration reduces the risk of hemorrhage, they have a longer half-life, and their platelet interactions are reduced in comparison to unfractionated heparin. They also provide an effective prophylaxis against postoperative major pulmonary embolism.

Oldenlandia

A plant genus of the family RUBIACEAE. Some species are used as an ingredient in Chinese and African traditional medicines. Members contain kalata B1, a macrocyclic peptide.

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