Category: Preclinical Development
Purpose: Berberine (BBR), jatrorrhizine (JAT), coptisine (COP) and palmatine (PAL) are key markers in Coptidis Rhizoma with BBR considered to be the main active components. It is already known that BBR not only has mechanistically and clinically been proven to be effective in anti-diabetic treatments but also has impact on compositions of gut microbiota. Our preliminary study also demonstrated significant degradation of BBR in rat intestinal content. Therefore, it is hypothesized that Coptidis Rhizoma extracts, containing BBR as major component, may also have potential effects on gut microbiota. In the current study, we proposed to 1) investigate the influence on BBR degradations by different Coptidis Rhizoma extracts in rat intestinal content; 2) test the feasibility of using such gut microbiota incubation method as a more differentiable quality control method of Coptidis Rhizoma extracts.
Methods: Three different extracts of Coptidis Rhizoma (Extracts A, B and C) were prepared by water extraction of Coptidis Rhizoma obtained from different resources. Male Sprague-Dawley (SD) rats (200-220 g) were sacrificed by excessive anesthesia followed by removal of intestine and immediate storage in the 0.9% cold saline solution. Subsequently, 2 g of the intestinal content was diluted by 18 ml 0.9% cold saline and homogenized to give a mixture of content suspension. All preparation procedures were maintained at 4℃ to keep the viability of bacteria for the incubation. To 950 µl intestinal content suspension of each rat, 50 µl of each extract (2 mg/ml in purified water) was spiked and incubated at 37℃ for 24-h. Samples were collected before (starting point) and 1, 2, 3, 6 and 24-h after the incubation followed by immediate storage in -20℃ to prevent further microbiota mediated metabolism. To 100 µl collected sample, 50 µl caffeine (CAF, internal standard) and 200 µl methanol with acetonitrile (1:1, v/v) were added to precipitate protein and then vortexed for 20 seconds at room temperature. After centrifugation at 13,000 rpm for 10 min, the supernatants were collected and injected to LC/MS/MS system for content analyses of BBR, JAT, COP and PAL. The liquid chromatographic separation was carried out on an Agilent Extend-C18 (2.1*250 mm, 5 μm) analytical column with mobile phase consisted of acetonitrile and 0.1% formic acid in water (30:70, v/v) running at a flow rate of 0.5 ml/min. For detecting concentration of each marker in Coptidis Rhizoma, quantification was carried out using selected reaction monitoring of m/z 336.1 → 320.1 for BBR, m/z 338.2 → 322.8 for JAT, m/z 320.1 → 292.1 for COP, m/z 352.2 → 336 for PAL, and m/z 195.1 → 138.0 for CAF.
Results: After Coptidis Rhizoma Extracts A, B and C incubation with rat intestinal contents for 24-h, among all the markers, BBR demonstrated to be the only component with significant degradations (80% remaining) (Figure 1). Meanwhile, comparing the degradation of BBR between different extracts suggested Extract C was significantly different from that of Extract A & B. were also found significant difference (p < 0.05) (Figure 2). Further content analyses of Extracts A, B & C revealed that total contents of JAT, COP and PAL in Extract C were significantly lower than that of Extracts A & B, whereas the BBR contents did not differ between the three extracts (Figure 3).
Conclusion: Although BBR contents in all three Coptidis Rhizoma extracts were similar, its gut-microbiota mediated degradation in Extract C significantly differed from the other two extracts. Since the activity of Coptidis Rhizoma extracts involved its alteration of gut microbiota, it is expected that the quality of Extract C may differ from the others.
Jiawen Shou– Hong Kong, Hong Kong
Pang Chui Shaw– Hong Kong, Hong Kong
Alice Pik Shan Kong– Division of Endocrinology, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, Hong Kong
Zhong Zuo– Director and Professor, School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, Hong Kong