Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 5:7:40278.
doi: 10.1038/srep40278.

Using the "target constituent removal combined with bioactivity assay" strategy to investigate the optimum arecoline content in charred areca nut

Wei Peng  1   2 , Yu-Jie Liu  1 , Mei-Bian Hu  1 , Dan Yan  1 , Yong-Xiang Gao  2 , Chun-Jie Wu  1
Affiliations

Using the "target constituent removal combined with bioactivity assay" strategy to investigate the optimum arecoline content in charred areca nut

Wei Peng et al. Sci Rep. .

Abstract

Charred areca nut (CAN) is used to treat dyspepsia and abdominal distension in children. However, reports revealed that arecoline, the most important active constituent of CAN, possesses potential toxicities. This study was designed to investigate the optimum arecoline content in CAN, using the "target constituent removal combined with bioactivity assay" strategy. Based on PTLC method, we prepared CAN lacking all arecoline (WAC-100R) and a series of different ratios of arecoline-removed CAN samples (WAC-Rx). MTT and acute toxicity assays indicated that decreasing content by 50% decreased CAN toxicity significantly. Animal results revealed arecoline contents over 50% could guarantee the beneficial effects of CAN on gastrointestinal tract. Additionally, decreasing arecoline content in CAN by 50% decreased its pro-apoptotic effects significantly. Furthermore, decreasing 50% arecoline content in CAN down-regulated the expressions of Cleaved-Caspase-3, c-jun, c-fos, COX-2, PGE2, and IL-1α. Thus, our results revealed that CAN with 50% arecoline content (WAC-50R) has similar beneficial effects on the gastrointestinal tract to CAN, whereas its toxicity was decreased significantly. Collectively, our study suggested that the strategy of "target constituent removal combined with bioactivity assay" is a promising method to identify the optimum arecoline content in CAN, which is approximately 0.12%.

PubMed Disclaimer

Figures

Figure 1
Figure 1. The strategy used to investigate the optimum arecoline content in charred areca nut.
Figure 2
Figure 2. Preparation of the arecoline removed totally charred areca nut samples (WAC-100R).
(A) Preparation of the WAC-100R samples by preparative thin layer chromatography (PTLC). 1 represents the WAC samples and the red band represented the arecoline, 2 shows the removal the arecoline band by PTLC, 3 shows the WAC-100R samples. (B) High performance liquid chromatography (HPLC) assay to determine the arecoline content of the WAC-100R samples. a–c represented the arecoline, WAC and WAC-100R, respectively.
Figure 3
Figure 3
Effects of different WAC-Rx samples on the contractility of stomach (A) and duodenum (B). The concentration of all the test samples was 40 μg/mL; **p < 0.01, *p < 0.05 vs. WAC.
Figure 4
Figure 4. Effects of WAC-Rx on gastric emptying, intestine promotion, and contents of motilin (MTL) and vasoactive intestinal peptide (VIP) in mice.
Mosapride (30 mg/kg) was used as the positive control drug. **p < 0.01, *p < 0.05 vs. control; #p < 0.05 vs. WAC.
Figure 5
Figure 5. Effects of WAC-Rx on gastric emptying, intestine promotion, motilin (MTL) and vasoactive intestinal peptide (VIP) contents in atropine sulfate-treated mice.
Mosapride (30 mg/kg) was used as the positive control drug. **p < 0.01, *p < 0.05 vs. control; #p < 0.05 vs. WAC
Figure 6
Figure 6. Pro-apoptotic effect of different WAC-Rx samples on HaCaT cells by 4′,6-diamidino-2-phenylindole (DAPI) staining.
HaCaT cells were treated with WAC-Nx at 300 μg/mL for 72 h, and the apoptotic cells were detected by DAPI staining and visualized under a fluorescent microscope (×200). (A–E) represented the control, WAC-100R, WAC-50R, WAC-30R, and WAC, respectively.
Figure 7
Figure 7. Pro-apoptotic effect of different WAC-Rx samples on HaCaT cells by flow cytometry.
HaCaT cells were treated with WAC-Rx at 300 μg/mL for 72 h, and the apoptotic cells were detected by staining with AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) followed by flow cytometry analysis. (A–E) represented the control, WAC-100R, WAC-50R, WAC-30R and WAC, respectively.
Figure 8
Figure 8. Effect of different WAC-Rx samples on the expressions of C-caspase-3, c-jun, and c-fos in HaCaT cells.
HaCaT cells were treated with WAC-Rx at 300 μg/mL for 72 h, and the expressions of C-caspase-3, c-jun, and c-fos in HaCaT cells were detected by western blotting. **p < 0.01, *p < 0.05 vs. control; ##p < 0.01, #p < 0.05 vs. WAC.
Figure 9
Figure 9. Effect of different WAC-Rx samples on the expressions of COX-2, PGE2, and IL-1α in HaCaT cells.
HaCaT cells were treated with WAC-Rx at 300 μg/mL for 72 h, and the expressions of COX-2, PGE2, and IL-1α in HaCaT cells were detected by western blotting. **p < 0.01, *p < 0.05 vs. control; ##p < 0.01, #p < 0.05 vs. WAC.
Figure 10
Figure 10. Flow chart of the preparation of CAN with arecoline totally removed (WAC-100R).
See this image and copyright information in PMC

References

    1. Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia (2015 edition) vol. 1, 365–366 (China Medical Science and Technology Press, China, 2010).
    1. Peng W. et al.. Areca catechu L. (Arecaceae): A review of its traditional uses, botany, phytochemistry, pharmacology and toxicology. J. Ethnopharmacol. 164, 340–356 (2015). - PubMed
    1. State Administration of Traditional Chinese Medicine. Chinese Material Medica, vol. 8, 439–648 (Science and Technology Press of Shanghai, China, 1999).
    1. Wang G. & Hu B. Research progress of arecoline. Int. J. Pathol. Clin. Med. 30, 171–175 (2010).
    1. Jiangsu New Medical College. Dictionary of Chinese Materia Medica, 2525–2528 (Science and Technology Press of Shanghai, China, 1977).

Publication types