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Figure Legends
Figue 1. HPLC analysis of MEMAC and compounds identification. (A) HPLC fingerprint of MEMAC. (B) Camphorataanhydride A (retention time = 27.13 min, peak 11) and camphorataimide B (retention time = 25.21 min, peak 8) were isolated and identified described as Materials and Methods section.
Figure. 2. Effect of MEMAC on cell viability of RAW264.7 and Human PBMCs.
RAW264.7 (2×105 cells/mL) cells and Human PBMCs (1×106 cells/mL) were treated with 0, 25, 50, 75, 100, 150 and 200 μg/mL of MEMAC for 24 h. Cell viability was checked after treatment with MEMAC using trypan blue exclusion method and examined using phase contrast microscopy. The results were displayed in percentage of control samples. In RAW264.7 cell experiment, data are presented as the mean ± S.D. for three independent experiments. Each data point represents the meant ± S.D. from one representative donor (n=3) in human PBMCs experiment; **p＜0.01, ***p＜0.001 as compared with the MEMAC treatment.
Figure 3. MEMAC inhibited immunogen induced pro-inflammatory cytokine and mediator production. RAW264.7 (2×105 cells/mL) cells were pretreated with MEMAC (0, 25, 50, and 75 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL), or other stimuli polyIC (20 μg/mL), Pam3 (Pam3CSK4, 200 ng/mL). Culture media were collected, the levels of (A) TNF-α and (B) IL-6 were measured at 18 h, the levels of (C) NO and (D) PGE2 were determined at 24 h. Data are presented as the mean ± S.D. for three independent experiments;
the same color column compare, *p＜0.05, **p＜0.01, ***p＜0.001 as compared with the LPS treatment.
Figure 4. MEMAC inhibited LPS-induced pro-inflammatory cytokine and mediator
production by human PBMCs. PBMCs (1×106 cells/mL) cells were pretreated with MEMAC (0, 25, 50, and 75 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL). Culture media were collected, the levels of (A) TNF-α and (B) IL-6 were measured at 18 h, the levels of (C) NO and (D) PGE2 were determined at 24 h. Each data point represents the meant ± S.D. from one representative donor (n=3); *p＜0.05, **p＜0.01, ***p＜0.001 as compared with the MEMAC treatment.
Figure 5. MEMAC suppressed LPS-induced COX-2 and iNOS expression. (A) Time-course effect. RAW264.7 cells were pretreated with MEMAC (75 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL) for 8 and 14 h. (B) Dose-dependent effect. RAW264.7 cells were treated with MEMAC (0, 10, 25, 50, and 75 μg/mL) for 30 min, and then stimulated with LPS (100 ng/mL) for another 24 h. Protein extract was analyzed by Western blotting using specific antibodies against COX-2 and iNOS. β-actin was used as an internal loading control.
Figure 6. Effects of MEMAC and indomethacin on hind paw edema induced by carrageenan in mice. Mice were i.p. treated with MEMAC (100, 200, and 400 mg/kg), indomethacin or normal saline, 30 min prior to subcutaneous injection of 1% -carrageenan (50 L) in the plantar side of right hind paws of the mice.
The paw volume was measured at 1, 2, 3, 4, and 5 h intervals after challenge using a plethysmometer. Each value was represented as mean ± S.D. *p < 0.05,
**p < 0.01 and ***p < 0.001 as compared with the carrageenan group (one-way
ANOVA followed by Scheffe’s multiple range test). Carr, carrageenan; Indo, indomethacin.
Figure 7. Effects of MEMAC and indomethacin on the levels of MDA, NO, and TNF-
production. Mice were i.p. treated with MEMAC (100, 200, and 400 mg/kg), indomethacin or normal saline, 30 min prior to injection of 1%
-carrageenan (50 L) in the plantar side of right hind paws of the mice.
After 5 h carrageenan challenge, the levels of (A) tissue MDA of hind paw in mice and the levels of (B) NO and (C) TNF-of serum in mice were measured. Each value represents as mean ± S.D. ###p < 0.001 as compared with the control group. *p < 0.05 and ***p < 0.001 as compared with the carrageenan group (one-way ANOVA followed by Scheffe’s multiple range test). Carr, carrageenan; Indo, indomethacin.
Figure 8. MEMAC alleviated carrageenan-induced hemorrhage, edema and leucocyte infiltration in mouse paw. (A) Mice were i.p. treated with MEMAC (100, 200, and 400 mg/kg), indomethacin or normal saline, 30 min prior to injection of 1% -carrageenan (50 L) in the plantar side of right hind paws of the mice.
After 5 h carrageenan challenge, the animals were sacrificed and the mouse right hind paw tissues were removed, the tissue slices were fixed and stained with hematoxylin and eosin (H&E) stain. All samples were observed and photographed with light microscopy. Carrageenan-treated mice showed
hemorrhage with moderately extravascular red blood cell and large amount of inflammatory leukocyte mainly neutrophils infiltration in the subdermis interstitial tissue. However, indomethacin (10 mg/kg) and MEMAC (50 mg/kg) significantly ameliorated carrageenan-induced pathological events (magnification, 100x). (B) Numbers of neutrophil infiltration in paw tissue was estimated (magnification, 400x). Each value was represented as mean ± S.D. ###p < 0.001 as compared with the control group. ***p < 0.001 as compared with carrageenan group. Scale bar, 100 μm. Carr, carrageenan; Indo, indomethacin.
Table 1: Effects of MEMAC, indomethacin, and carrageenan on the liver catalase, superoxide dismutase, and glutathione peroxidase activities in mice.
Groups Catalase
(U/mg protein)
Superoxide dismutase (U/mg protein)
Glutathione peroxidase (U/mg protein)
Control 4.87 ± 0.06 26.21 ± 0.26 2.56 ± 0.32
Carr 3.13 ± 0.42### 16.45 ± 0.22### 1.42 ± 0.08###
Carr + Indo 3.95 ± 0.37** 20.25 ± 0.34** 2.15 ± 0.19*
Carr + MEMAC (5 mg/Kg) 3.51 ± 0.23* 19.56 ± 0.17* 1.54 ± 0.07 Carr + MEMAC (25 mg/Kg) 3.98 ± 0.21** 23.46 ± 0.27** 1.83 ± 0.16* Carr + MEMAC (50 mg/Kg) 4.28 ± 0.32*** 24.89 ± 0.12*** 2.34 ± 0.12**
Each value was represented as mean ± S.D. ###p < 0.001 as compared with the control group. *p <
0.05, **p < 0.01, ***p < 0.001 as compared with the carrageenan group (one-way ANOVA followed by Scheffe’s multiple range test). Carr, carrageenan; Indo, indomethacin.
Figure. 1.
Figure. 2.
Figure.3
Figure 4
Figure 5
Figure 6
Time (h)
0 1 2 3 4 5
Changes of edema volume (mL)
0.00 0.02 0.04 0.06 0.08
Carr
Carr and Indo
Carr and MEMAC 5 mg/kg Carr and MEMAC 25 mg/kg Carr and MEMAC 50 mg/kg
*** ***
** **
**
* *
Figure 7
Figure 8
B.
***
Control - Indo 50 MEMAC (mg/kg)
** **
Neutrophil / scope (cell)
0 20 40 60 80 100
***
***
Carr
###