In order to clarify the physiological effects of Chlorella intake on subjects with high-risk factors for lifestyle-related diseases, we conducted Chlorella ingestion tests on 17 subjects with high-risk factors for lifestyle-related diseases and 17 healthy subjects over a 16-week period, including a 4-week post-observation period. We conducted blood biochemical tests and analyzed gene expression profile in whole blood cells in the peripheral blood before and after Chlorella intake. We confirmed that in both groups, Chlorella intake resulted in noticeable reductions in body fat percentage, serum total cholesterol, and fasting blood glucose levels. Through gene expression analysis, we found that gene expression profiles varied with Chlorella intake and identified many genes that exhibited behavior such that after the completion of the intake period, expression levels returned to pre-intake expression ones. Among these were genes related to signal transduction molecules, metabolic enzymes, receptors, transporters, and cytokines. A difference in expression level was found between the two groups at the start of the tests, and we were able to identify genes with noticeable variance in expression level resulting from Chlorella intake in the high-risk factor group. These included genes involved in fat metabolism and insulin signaling pathways, which suggests that these pathways could be physiologically affected by Chlorella intake. There were clear variations in the expression profiles of genes directly related to uptake of glucose resulting from Chlorella intake, indicating that the activation of insulin signaling pathways could be the reason for the hypoglycemic effects of Chlorella.

Vitamin B was determined and characterized in 19 dried Chlorella health supplements. Vitamin contents of dried Chlorella cells varied from <0.1 μg to approximately 415 μg per 100 g of dry weight. Subsequent liquid chromatography/electrospray ionization-tandem mass spectrometry analyses showed the presence of inactive corrinoid compounds, a cobalt-free corrinoid, and 5-methoxybenzimidazolyl cyanocobamide (factor IIIm) in four and three high vitamin B-containing Chlorella tablets, respectively. In four Chlorella tablet types with high and moderate vitamin B contents, the coenzyme forms of vitamin B 5'-deoxyadenosylcobalamin (approximately 32%) and methylcobalamin (approximately 8%) were considerably present, whereas the unnaturally occurring corrinoid cyanocobalamin was present at the lowest concentrations. The species Chlorella sorokiniana (formerly Chlorella pyrenoidosa) is commonly used in dietary supplements and did not show an absolute requirement of vitamin B for growth despite vitamin B uptake from the medium being observed. In further experiments, vitamin B-dependent methylmalonyl-CoA mutase and methionine synthase activities were detected in cell homogenates. In particular, methionine synthase activity was significantly increased following the addition of vitamin B to the medium. These results suggest that vitamin B contents of Chlorella tablets reflect the presence of vitamin B-generating organic ingredients in the medium or the concomitant growth of vitamin B-synthesizing bacteria under open culture conditions.

A Chlorella powder was screened using 52 in vitro assay systems for enzyme activity, receptor binding, cellular cytokine release, and B and T cell proliferation. The screening revealed a very potent inhibition of human protein tyrosine phosphatase (PTP) activity of CD45 and PTP1C with 50% inhibitory concentration (IC(50)) values of 0.678 and 1.56 microg/mL, respectively. It also showed a moderate inhibition of other PTPs, including PTP1B (IC(50) = 65.3 microg/mL) and T-cell-PTP (114 microg/mL). Other inhibitory activities and their IC(50) values included inhibition of the human matrix metalloproteinases (MMPs) MMP-1 (127 microg/mL), MMP-3 (185 microg/mL), MMP-7 (18.1 microg/mL), and MMP-9 (237 microg/mL) and the human peptidase caspases caspase 1 (300 microg/mL), caspase 3 (203 microg/mL), caspase 6 (301 microg/mL), caspase 7 (291 microg/mL), and caspase 8 (261 microg/mL), as well as release of the cytokines interleukin (IL)-1 (44.9 microg/mL), IL-2 (14.8 microg/mL), IL-4 (49.2 microg/mL), IL-6 (34.7 microg/mL), interferon-gamma (31.6 microg/mL), and tumor necrosis factor-alpha (11 microg/mL) from human peripheral blood mononuclear cells. Chlorella also inhibited B cell proliferation (16.6 microg/mL) in mouse splenocytes and T cell proliferation (54.2 microg/mL) in mouse thymocytes. The binding of a phorbol ester, phorbol 12,13-dibutyrate, to its receptors was also inhibited by Chlorella with an IC(50) of 152 microg/mL. These results reveal potential pharmacological activities that, if confirmed by in vivo studies, might be exploited for the prevention or treatment of several serious pathologies, including inflammatory disease and cancer.

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