Fig 1.
Concentration of major components and free radical scavenging activity of GEGR.
(A) The distribution of three major components of GEGR, gallotannin, gallic acid and methylgallate, was analyzed based on their UV-Vis spectra. (B) HPLC chromatograms of pure gallic acid (commercial chemical), pure methylgallate (commercial chemical), pure gallotannin (commercial chemical) and GR extract. (C) DPPH radical scavenging activity of GEGR. The data shown represent the means ± SD of three replicates.
Table 1.
Alteration of feeding behavior and constipation parameters in the constipated SD rats after GEGR treatment.
Fig 2.
Alteration of histological structure in Lop-induced constipated rats.
H&E stained sections of transverse colon rats from the No treated group, GEGR treated group, Lop+vehicle treated group, Lop+GEGR treated group or Lop+BS treated group were observed at 100× (upper corner in left column) and 200× (left column) using a light microscope. Mucin secreted from crypt layer cells was stained with alcian blue at pH 2.5, and their structures were observed at 200× (right column). Five to six rats per group were assayed in triplicate by H&E and alcian blue staining.
Table 2.
Histopathological alteration of the constipated SD rats after GEGR treatment.
Fig 3.
Expression of mAChRs transcript in the transverse colon.
The levels of mAChR M2 and M3 transcripts in the total mRNA of transverse colons were measured by RT-PCR using specific primers. After the intensity of each band was determined using an imaging densitometer, the relative levels of mAChR M2 and M3 transcripts were calculated based on the intensity of actin transcripts. Five to six rats per group were assayed in triplicate by RT-PCR assays. Data represent the means±SD of three replicates. a, p<0.05 compared to the No treated group. b, p<0.05 compared to the Lop+vehicle treated group.
Fig 4.
Expression of key proteins in the mAChR M2 and M3 signaling pathway.
The expression of several related proteins in the mAChR M2 and M3 signaling pathway was measured by Western blot analysis using HRP-labeled anti-rabbit IgG antibody. After the intensity of each band was determined using an imaging densitometer, the relative levels of six proteins were calculated based on the intensity of actin protein. Five to six rats per group were assayed in triplicate by Western blotting. Data represent the means ± SD of three replicates. a, p<0.05 compared to the No treated group. b, p<0.05 compared to the Lop+vehicle treated group.
Fig 5.
Alteration of the expression of Gα proteins and IP3 concentration.
(A) The expression of Gα within G protein signaling was measured by Western blotting using HRP-labeled anti-rabbit IgG antibody. After the intensity of each band was determined using an imaging densitometer, the relative levels of protein were calculated based on the intensity of actin protein. (B) The tissue homogenate used to measure the IP3 concentration was prepared from transverse colons collected from SD rats of subset groups. The IP3 concentration was quantified by an enzyme-linked immunosorbent assay with a sensitivity of 0.39 ng/ml and an inter-assay coefficient of variation of 1.56–100 ng/ml. Data shown are the means ± SD (n = 5). a, p<0.05 compared to the No treated group. b, p<0.05 compared to the Lop+vehicle treated group.