Protective Effect of Ethanolic Extract of Djulis Hull on Indomethacin-Induced Gastric Injury
, Nai-Chen Liao 4, Hui-Yun Tsai 5,6, Jue-Liang Hsu 2, Pi-Jen Tsai 4, Agustin Krisna Wardani 1 and Yu-Kuo Chen 4,*Round 1
Reviewer 1 Report
In the present paper by Isnain et al., the ethanolic extract of djulis hull (EEDH) was investigated for its gastroprotective effect on indomethacin (IND)-induced gastric damage in C57BL/6J mice. Results of the study demonstrated that pretreatment with EEDH significantly alleviated the gastric ulcer caused by IND in a dose-dependent manner. Mechanistic studies indicated that the protective effect of EEDH was associated with modulation of antioxidant status and decreases in inflammatory mediators in gastric mucosa. These novel findings are interesting and the study was technically well performed. Some minor concerns:
1. Why were 120 mg/kg IND, 200 and 500 mg/kg EEDH chosen for the experiment? Please discuss the rationale of dose selection in the experiment.
2. Since more than two experimental groups were included in the study, ANOVA would be the appropriate statistic method to determine the differences among the groups.
3. Please add arrows in Figure 1 to indicate the areas of ulcer in the gastric mucosa.
Author Response
Reviewer: 1
Comments:
In the present paper by Isnain et al., the ethanolic extract of djulis hull (EEDH) was investigated for its gastroprotective effect on indomethacin (IND)-induced gastric damage in C57BL/6J mice. Results of the study demonstrated that pretreatment with EEDH significantly alleviated the gastric ulcer caused by IND in a dose-dependent manner. Mechanistic studies indicated that the protective effect of EEDH was associated with modulation of antioxidant status and decreases in inflammatory mediators in gastric mucosa. These novel findings are interesting and the study was technically well performed. Some minor concerns:
- Why were 120 mg/kg IND, 200 and 500 mg/kg EEDH chosen for the experiment? Please discuss the rationale of dose selection in the experiment.
Response: Thanks for reviewer's suggestion. In our preliminary study (n=3 each group), we found that 120 mg/kg IND treatment caused appropriate gastric ulcer area in C57/BL mice, and preadministration of 200 mg/kg EEDH and 200 mg/kg water extract of djulis hull (WEDH) improved the gastric damage caused by IND. Among them, EEDH revealed better protective activity. That is why we chose 120 mg/kg IND, 200 and 500 mg/kg EEDH for subsequent experiment to elucidate whether a 2.5-fold higher dose of EEDH shows greater gastric gastroprotective effect. We have added this statement in the section of Discussion (the third paragraph).
- Since more than two experimental groups were included in the study, ANOVA would be the appropriate statistic method to determine the differences among the groups.
Response: Thanks for reviewer's suggestion. ANOVA was used as the statistic method to replace the original method in this study, and we obtained same statistic results. Therefore, we used same symbol to indicate the difference among the groups. However, we revised the statistic method in Section 2.9.
- Please add arrows in Figure 1 to indicate the areas of ulcer in the gastric mucosa.
Response: Thanks for reviewer's suggestion. The arrows were added in Figure 1 to indicate the areas of ulcer in the gastric mucosa.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In the present study titled “Protective effect of ethanolic extract of djulis hull on indomethacin-induced gastric injury” by Isnain et al., the authors used an indomethacin (IND)-induced gastric damage model to investigate the gastroprotective effect of ethanolic extract of djulis hull (EEDH) in mice. They found that EEDH dose-dependently attenuated the gastric ulcer caused by IND via restoration of antioxidant status in gastric mucosa of mice, including the level of GSH and the activities of SOD and CAT. Moreover, the results of this study also showed that IND-induced protein expression of inflammatory mediators (TNF-α and iNOS) of gastric mucosa in mice was downregulated by pretreatment of EEDH. Basically, this study is well done and worthy of publication in this journal. However, there are still a few issues with this research article (as follows). I would recommend the authors revise the manuscript accordingly:
1. Why did the authors choose the alcoholic extract of djulis hull as the experimental sample? Have the authors tried other solvent extracts (such as aqueous extract)?
2. It seems clear that this study did not aim at developing a new treatment for gastric ulcer of mice, but to provide new tracks for the human disease. They indeed claimed that EEDH can be developed as a functional food in the future. Therefore, the authors should discuss the possible human equivalent of 500 mg/kg used in mice.
3. If possible, please provide the original blot of the WB analysis results.
4. Indicate the extraction temperature to obtain the EEDH (lines 106-107).
5. A hyphen is missing between Tween and 20 (line 89).
6. There is an extra closing parenthesis for (NH4)2S2O8 (line 90).
7. It looks like there is an extra space in front of RIPA (line 90).
8. "3." should be corrected to "(3)" (line 116).
9. When referring to IUCUC for the first time, it is recommended that its full name be listed (line 122).
Comments for author File: 
Comments.docx
Author Response
Reviewer: 2
Comments:
In the present study titled “Protective effect of ethanolic extract of djulis hull on indomethacin-induced gastric injury” by Isnain et al., the authors used an indomethacin (IND)-induced gastric damage model to investigate the gastroprotective effect of ethanolic extract of djulis hull (EEDH) in mice. They found that EEDH dose-dependently attenuated the gastric ulcer caused by IND via restoration of antioxidant status in gastric mucosa of mice, including the level of GSH and the activities of SOD and CAT. Moreover, the results of this study also showed that IND-induced protein expression of inflammatory mediators (TNF-α and iNOS) of gastric mucosa in mice was downregulated by pretreatment of EEDH. Basically, this study is well done and worthy of publication in this journal. However, there are still a few issues with this research article (as follows). I would recommend the authors revise the manuscript accordingly:
- Why did the authors choose the alcoholic extract of djulis hull as the experimental sample? Have the authors tried other solvent extracts (such as aqueous extract)?
Response: Thanks for reviewer's question. In our preliminary study (n=3 each group), we found that preadministration of 200 mg/kg EEDH and 200 mg/kg water extract of djulis hull (WEDH) improved the gastric damage caused by IND. Among them, EEDH revealed better protective activity. That is why we chose 200 and 500 mg/kg EEDH for subsequent experiment to elucidate whether a 2.5-fold higher dose of EEDH shows greater gastric gastroprotective effect. We have added this statement in the section of Discussion (the third paragraph).
- It seems clear that this study did not aim at developing a new treatment for gastric ulcer of mice, but to provide new tracks for the human disease. They indeed claimed that EEDH can be developed as a functional food in the future. Therefore, the authors should discuss the possible human equivalent of 500 mg/kg used in mice.
Response: Thanks for reviewer's suggestion. The discussion for the possibility and feasibility of using the human equivalent of 200-500 mg/kg in mice was addressed in the section of Discussion (penultimate sentence of the last paragraph).
- If possible, please provide the original blot of the WB analysis results.
Response: Thanks for reviewer's suggestion. We have provided the original blot image for the WB analysis results.
- Indicate the extraction temperature to obtain the EEDH (lines 106-107).
Response: Thanks for reviewer's suggestion. We have added the extraction temperature (at room temperature) to obtain the EEDH in the section 2.2.
- A hyphen is missing between Tween and 20 (line 89).
Response: Thanks for reviewer's reminder. We have revised it in the section 2.1.
- There is an extra closing parenthesis for (NH4)2S2O8 (line 90).
Response: Thanks for reviewer's reminder. We have revised it in the section 2.1.
- It looks like there is an extra space in front of RIPA (line 90).
Response: Thanks for reviewer's reminder. We have revised it in the section 2.1.
- "3." should be corrected to "(3)" (line 116).
Response: Thanks for reviewer's reminder. We have revised it in the section 2.3.
- When referring to IUCUC for the first time, it is recommended that its full name be listed (line 122).
Response: Thanks for reviewer's reminder. We have revised it in the section 2.3.
Author Response File: Author Response.pdf
