Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 1057, Issue -, Pages 70-80Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2017.05.004
Keywords
Intestinal bacteria; In vitro fermentation; Short-chain fatty acids; Gas chromograph; Berberine
Funding
- National Health and Family Planning Commission of the PRC Program [201402001]
- Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD study [Z141102004414062]
- CAMS Major Collaborative Innovation Project [2016-I2M-1-011]
- Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study [BZ0150]
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Short-chain fatty acids (SCFAs) of intestine microbial have caught accumulating attention for their beneficial effects on human health. Botanic compounds with low bioavailability such as berberine (BBR) and resveratrol might interact with intestinal microbial ecosystem and promote gut bacteria to produce SCFA, which contribute to their biological effects. In the present study, a comprehensive assay system was built to detect SCFAs production in intestinal bacteria, in which stringent anaerobic culture was applied for in vitro bacterial fermentation, followed by direct-injection GC detection (chemical detection) in combination with real time polymerase chain reaction (RT-PCR, biological detection). BBR was used as positive reference. The direct injection GC method was calibrated and successfully applied to analyze the concentration of SCFAs in gut microbiota and BBR was proved to be effective in the dose- and time-dependent up-regulation of SCFAs production. As compared to the saline group, the concentration of acetic acid, propionate acid and butyric acid (the main SCFAs in gut microbiota) were increased by 17.7%, 11.1% and 30.5%, respectively, after incubating intestinal bacteria with 20 mu g/mL BBR for 24 h. The increase reached to 34.9%, 22.4% and 51.6%, respectively when the BBR was 50 mu g/mL. Additionally, consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) were designed for the detection of acetate kinase (ACK), Methyhnalonyl-CoA decarboxylase (MMD) and butyrylCoA: acetate-CoA transferase (BUT), as they are the key enzymes in the synthetic pathway for acetic acid, propionate acid and butyric acid, respectively. After 24hr's incubation, BBR was shown to promote the gene expression of ACK, MMD and BUT significantly (86.5%, 27.2% and 60.4%, respectively, with 20 mu g/mL BBR; 130.2%, 84.2% and 98.4%, respectively, with 50 mu g/mL BBR), showing a solid biological support for the chemical detection. This comprehensive assay system might be useful in identifying SCFAs promoting agents with information on their mechanism.
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