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Journal of Chinese Integrative Medicine ›› 2010, Vol. 8 ›› Issue (6): 554-561.doi: 10.3736/jcim20100608

• Original Experimental Research • Previous Articles     Next Articles

Effects of extract of Polygonum multiflorum on cell cycle arrest and apoptosis of human liver cell line L02

Rui-chen Zhanga,Bin Liub,Zhen-xiao Suna,Dong-yan Xub   

  1. a Department of Biopharmaceutics, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China
    b Department of Traditional Chinese Medicine Chemistry, School of Chinese Materia Medica, Beijing University of Chinese Medicine,Beijing 100102,China
  • Received:2010-03-01 Accepted:2010-04-12 Online:2010-06-20 Published:2010-06-15
  • Contact: Zhen-xiao Sun E-mail:sunzxcn@hotmail.com

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Objective

To analyze the chemical constituents of Polygonum multiflorum extract which may cause human liver cell damage and to explore the mechanism.
Methods

Raw and processed Polygonum multiflorum were extracted by 70% ethanol, then raw and processed Polygonum multiflorum water-eluted material (RW and PW), 50% ethanol-eluted material (R50 and P50) and 95% ethanol-eluted material (R95 and P95) were obtained by absorbing through AB-8 macroporous resin, followed by water, 50% ethanol and 95% ethanol elution in order. The water extracts of raw and processed Polygonum multiflorum (RWE or PWE) were obtained by boiling them in water as usual. Normal human liver L02 cells were treated by different concentrations of eluted Polygonum multiflorum materials for different time, and the cell growth inhibition of each group was determined by methylthiazolyldiphenyl-tetrazolium bromide method. The chemical constituents which had a significant cytotoxicity to L02 cells were analyzed by high-performance liquid chromatography (HPLC). Morphological changes of L02 cells were observed by Giemsa staining and cell cycle distribution was observed by flow cytometry.
Results

It was found that 95% ethanol-eluted extracts of raw and processed Polygonum multiflorum showed significant growth inhibition on normal human liver L02 cells, while the other components showed no significant inhibition on cell growth. HPLC analysis showed that the main component in 95% ethanol-eluted extract of raw and processed Polygonum multiflorum was emodin at content of (18.53±2.96)% and (10.28±1.34)% respectively. Cell cycle analysis showed that 95% ethanol-eluted material of Polygonum multiflorum and emodin had a similar significant effect of S phase arrest and all could induce L02 cell apoptosis.
Conclusion

The main part of Polygonum multiflorum causing liver cell damage is the 95% ethanol-eluted extract, and emodin is one of the important chemical constituents leading to liver cell damage.

Key words: Polygonum multiflorum, Hepatocytes, Cytotoxicity, Liver damage, Chemical constituents, Anthra- quinone, Emodin, Cell cycle

"

Figure 1

Components of R95 and P95 observed by HPLCA: Reference substances; B: R95; C: P95. ①: Aloe-emodin; ②: Rhein; ③: Emodin; ④: Chrysophanol; ⑤: Physcion."

"

Figure 2

Morphological changes of L02 cells after 48-h culture in different groups (Inverted microscopy, ×200) A: Control group; B: 100 μg/mL R95 group; C: 100 μg/mL P95 group; D: 50 μg/mL emodin group. Cells in group A show adjacent cells connecting, all in good shape with uniform refraction of cytoplasm and obvious nucleus. Cells in group B, C and D show margination of nuclear substances around the nuclear envelope and some vacuole-like structure appearing around the nucleus(→), cells shrinking from the surrounding cells(△), cell apoptosis and apoptotic bodies appearing(▲)."

Figure 3

Morphological changes of L02 cells after 48-h culture in different groups observed by Giemsa staining (Inverted microscopy, ×200) A: Control group; B: 100 μg/mL R95 group; C: 100 μg/mL P95 group; D: 50 μg/mL emodin group. Cells are intact in group A with large and clear outline of nucleus, and uniform cytoplasm. Morphology of some L02 cells and their nucleus is abnormal in group B, C and D, showing chromatin margination (→), apoptotic cells(△) and apoptotic bodies(▲)."

Figure 4

Cell cycle distributions of L02 cells after R95, P95 and emodin treatment for 0, 24 and 48 h detected by flow cytometry"

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