Antileukemic effects of indigo naturalis constituents by "target constituent knock-out" coupled with semipreparative liquid chromatography and quadrupole time-of-flight mass spectrometry.

A novel approach is presented to identify constituents with antileukemic properties in extracts of Indigo naturalis (Qingdai in Chinese). Target compounds (A+ , BC+ , and ABC+ ) that knocked out specific constituents displayed antileukemic effects in a total extract of I. naturalis and negative constituents (A- , BC- , and ABC- ) that knocked out target compounds were separated, identified and knocked out by semipreparative liquid chromatography (semipreparative HPLC) and quadrupole time-of-flight mass spectrometer. Quantitative methods were used to evaluate the content of each knocked-out constituent in the total extract (D). Subsequently, interactions between the antileukemic effects of knocked-out constituents and D were screened and evaluated at the cellular level. Negative constituents including A- (65.47% ± 1.20%), BC- (54.61% ± 2.43%) and ABC- (67.49% ± 3.28%) displayed a greater inhibitory effect than D (47.16% ± 0.072%), which was not knocked out after 24 h of incubation, whereas the target compounds had not superior. Target compounds may have caused an antagonistic effect on the corresponding negative constituents. After 48 h, inhibition of proliferation by D (75.48% ± 3.78%) increased compared with that by negative constituents, whereas the antagonistic effect of target components on negative constituents was diminished. This result may reflect competitive antagonism. Comparing the reactions after 24 and 48 h, the inhibitory ratio of ABC- (79.29% ± 1.22%) in these knocked-out constituents and D was always the highest. With different concentrations tested after 48 h, ABC- significantly increased the rate of apoptosis on K562 cells (P < 0.01), indicating that in addition to indirubin, tryptanthrin and isorhamnetin, other antileukemic constituents may be present. Our study presents an approach that is a truer reflection of the antileukemic effects of knocked-out constituents in I. naturalis supported by reference to pharmacodynamic actions and the quality of I. naturalis. The approach may be useful for the analysis of other herbal extracts found in traditional Chinese medicine.

The endogenous development and pathogenicity of Eimeria anseris (Kotlan, 1932) in domestic goslings.

Twenty-one, 25-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 0.5 × 104, 1 × 104, or 100 × 104 sporulated oocysts of Eimeria anseris and sacrificed at intervals from 24 to 216 h post-inoculation (HPI). Nine uninfected goslings served as negative controls. Parts of the visceral organs from goslings, including the intestines, kidneys, and liver, were fixed, sectioned, and observed microscopically. The results revealed that two generations of meronts occurred in the life cycle of E. anseris. The first generation of meronts developed at 24-96 HPI and the second generation at 90-128 HPI. Each meront contained 4-10 merozoites. Development of gamonts began at 128 HPI and mature oocysts appeared at 168 HPI. Developmental stages presented mainly in the epithelial cells of crypts and lamina propria in the posterior parts of the jejunum and ileum. Parasites localized mostly in the cytoplasm and occasionally in the nuclei of host cells. Histological lesions were pronounced in the jejunum and ileum. Desquamation and necrosis of the epithelium of intestine and crypts, infiltration of inflammatory cells, and hemorrhage and mucosal edema were associated with aggregates of endogenous stages. The infected goslings mainly showed severe diarrhea, depression, anorexia, and emaciation, suggesting that E. anseris is highly pathogenic in goslings.