Antihypertensive Effects of Polyphenolic Extract from Korean Red Pine (Pinus densiflora Sieb. et Zucc.) Bark in Spontaneously Hypertensive Rats

Round 1

Reviewer 1 Report

The authors of this manuscript have investigated the antihypertensive property of red pine bark extract from korea. They have confirmed the presence of polyphenolics in the extract by HPLC and claim that the antihypertensive activity is related to the antioxidant activity of the polyphenolics. I have few minor changes and two major changes as below.

Minor changes:

Line 23: Every once a week should be :Once every week

Line 24: no need of ‘these’

Line 29: those of the SHR control group: those ‘in’ the SHR control group

Line 39: full stop is missing after the sentence.

Line 46: needs ‘has’  in the sentence

Line 55: important to the treatment of hypertension: important ‘for’ the treatment of hypertension

Line 54: The research suggested that scavenging ROS is also important to the treatment of hypertension, and that ACEI with antioxidative activity such as phenolic compounds is a preferable antihypertensive agent. This sentence needs reconstruction.

Line 64: Our previously study: previous

Line 66: strong antioxidative effects: strong antioxidative ‘properties’

Line 107: at the same time of day: at same time of the day

Line 120: tissues from lungs and kidneys: lungs and kidneys are tissues

Line 121: 5.7 mM of HHL: how much volume of HHL was added to each well?

Line 125: Fifteen microliters: 15uL

Line 126: After incubation: after shaking?

Figure 1 has only one graph repeated two times.

There no need of full forms of the pine extract or the type of rat in the figure captions.

Line 278: no need of dashes in five-week-old

Line 282: no need of ‘thus’

Line 295: RAS thus, ACEIs are mainly : full stop after RAS

Line 303: two SHR groups fed KRPBE : two SHR groups fed with KRPBE

Major changes: 

The authors have nicely explained what causes the high blood pressure: Pathophysiology of hypertension in SHR is due to overall changes in RAS components, such as renin 280 activity, ACE activity, angiotensin II content, AT1 receptor expression, and NADPH oxidase activity. But if they also discuss why there are these changes in the RAS components, it would be helpful in the discussion.

Discussion could be rewritten without mentioning the figure numbers again, as those are given in the results section.

Author Response

The authors of this manuscript have investigated the antihypertensive property of red pine bark extract from korea. They have confirmed the presence of polyphenolics in the extract by HPLC and claim that the antihypertensive activity is related to the antioxidant activity of the polyphenolics. I have few minor changes and two major changes as below.

We aprreciate to reviewer's valuable comments.

Minor changes:

Line 23: Every once a week should be :Once every week

Revised accordingly (line 23).

Line 24: no need of ‘these’

Revised accordingly (line 23).

Line 29: those of the SHR control group: those ‘in’ the SHR control group

Revised accordingly (line 28).

Line 39: full stop is missing after the sentence.

Revised accordingly (line 38).

Line 46: needs ‘has’  in the sentence

Revised accordingly (line 45).

Line 55: important to the treatment of hypertension: important ‘for’ the treatment of hypertension

Revised accordingly (line 54).

Line 54: The research suggested that scavenging ROS is also important to the treatment of hypertension, and that ACEI with antioxidative activity such as phenolic compounds is a preferable antihypertensive agent. This sentence needs reconstruction.

Revised accordingly (Lines 54-55).

Line 64: Our previously study: previous

Revised accordingly (line 63).

Line 66: strong antioxidative effects: strong antioxidative ‘properties’

Revised accordingly (line 65).

Line 107: at the same time of day: at same time of the day

These two mean the same thing. The phrase “at the same time of day” was modified into “at the same time of the day” (line 121).

Line 120: tissues from lungs and kidneys: lungs and kidneys are tissues

The phrase “serum and tissues from lungs and kidneys” was revised into “serum, lungs, and kidneys” (line 135).

Line 121: 5.7 mM of HHL: how much volume of HHL was added to each well?

Thirty microliters of 5.7 mM of HHL was added. Revised accordingly (line 136).

Line 125: Fifteen microliters: 15uL

Sentences are not supposed to start with a number. Therefore, we will leave it the same (line 140).

Line 126: After incubation: after shaking?

Thank you for your suggestion. We just changed it to “Then” (line 141).

Figure 1 has only one graph repeated two times.

There no need of full forms of the pine extract or the type of rat in the figure captions.

Thank you for your considerable comments. We revised all the figure captions including Figure 1.

Line 278: no need of dashes in five-week-old

Revised to five weeks (line 270).

Line 282: no need of ‘thus’

Revised accordingly (line 277).

Line 295: RAS thus, ACEIs are mainly : full stop after RAS

Revised accordingly (line 290).

Line 303: two SHR groups fed KRPBE : two SHR groups fed with KRPBE

Revised accordingly (line 298).

The authors have nicely explained what causes the high blood pressure: Pathophysiology of hypertension in SHR is due to overall changes in RAS components, such as renin 280 activity, ACE activity, angiotensin II content, AT1 receptor expression, and NADPH oxidase activity. But if they also discuss why there are these changes in the RAS components, it would be helpful in the discussion.

Discussion could be rewritten without mentioning the figure numbers again, as those are given in the results section.

A new discussion “why there are these changes in the RAS components” was added (Line 273-277). The figures numbers were deleted according to reviewer’s comment.

Reviewer 2 Report

Comments:

• Since authors used to extract from the bark, how did they ensure that the percentage of various polyphenols remain the same from batch to batch preparation?
• Since authors used bark to extract, how did they eliminate lignin a complex polyphenol from the extract?
• In the result section, authors used Fig.1A side by side instead of 1B. However, they describe about 1B in the legend. They should rectify this mistake.
• Authors used SHR in their study. Same rat model was used to study the effect of NaCl on blood pressure. Others have shown that NaCl (common salt) induced BP via g-MSH. So what steps authors have taken to decrease the influence of NaCl in the rat food or do they have an explanation for using regular diet for SHR in their experiment
• Even though authors used KRPBE at two concentrations 50 mg and 150 mg, except in lung ACE activity assay, in all other assays the results were similar. Though they used 3X concentration, KRPBE did not show a dose-dependent effect, why?
• Though authors mention about AT1 receptor expression in their discussion, they have not done any assay to monitor AT1 receptor expression in their study, Why?
• Use of Ethyl ether as an anesthetic is obsolete, now-a-days scientists use isofluorene, Ketamine as anesthetic agents. So they should follow this in their future work

Author Response

Thank you for reviewer's considerable comments.

The attached word file contains the response.

Author Response File: Author Response.docx

Reviewer 3 Report

The antihypertensive effect of a natural phenol extract is studied in a rat model in this very interesting article. The experimental design is correct, the methodology used is adequate and the results are clear and conveniently presented.

Only some minor suggestions:

Figure 1: figure 1B is missing instead figure 1A is repeated.

Page 6, lines 243,244: If the difference between SHR+CAP and SHR groups are not significant they should be considered equals and therefore I would suggest not to say that SHR+CAP MAD content is lower than that of SHR group.

I would suggest using Nitric oxide instead of NO all over the text.

Author Response

The antihypertensive effect of a natural phenol extract is studied in a rat model in this very interesting article. The experimental design is correct, the methodology used is adequate and the results are clear and conveniently presented.

We appreciate reviewer's valuable comments.

Only some minor suggestions:

Figure 1: figure 1B is missing instead figure 1A is repeated.

We made a mistake in copying and pasting after editing of Figure 1B. Figure 1B was added in manuscript.

Page 6, lines 243,244: If the difference between SHR+CAP and SHR groups are not significant they should be considered equals and therefore I would suggest not to say that SHR+CAP MAD content is lower than that of SHR group.

Reviewer4 suggested to change statistical analysis form student’s t-test to ANOVA followed by post-hoc test. Tukey–Kramer honestly significant difference test showed that lung MDA content of SHR+CAP was significantly different from that of SHR group. Anyhow, the sentence was concisely revised (lines 243-245).

I would suggest using Nitric oxide instead of NO all over the text.

All "NO” in manuscript have been changed to Nitric oxide.

Reviewer 4 Report

The manuscript presents the interesting study. The study was well designed and written. I have only a few issues: 1. Statistical analysis: in my opinion Authors should used ANOVA test because they analysed five groups; it should be changed. 2. Phenolics in KRPBE should be analysed before the animal study. Quantification of phenolics should be described at the beginning of the Materials and methods part and Results part. 3. Fig 5 should be moved to supplementary materials. 4. Conclusions: The sentence: “Our study suggests that KRPBE rich in(….) “ page 10, lines 370-371 is too far reaching and should be removed or redescribed; Based on this results it is hard to say if KRPBE is safe for hypertensive patients and the effectiveness of this extract must be confirmed in clinical trials.

Author Response

The manuscript presents the interesting study. The study was well designed and written.

We appreciate to reviewer's valuable comments.

I have only a few issues:

1. Statistical analysis: in my opinion Authors should used ANOVA test because they analysed five groups; it should be changed.

We changed the statistical analysis from Student’s t-test to ANOVA followed by Tukey–Kramer honestly significant difference test.

2. Phenolics in KRPBE should be analysed before the animal study. Quantification of phenolics should be described at the beginning of the Materials and methods part and Results part.

Revised accordingly (2.2. Quantification of phenolics using HPLC in the Materials and methods section; 3.1. Quantification of phenolics using HPLC in the Results section).

3. Fig 5 should be moved to supplementary materials.

Revised accordingly (Supplementary Figure 1).

4. Conclusions: The sentence: “Our study suggests that KRPBE rich in(….) “ page 10, lines 370-371 is too far reaching and should be removed or redescribed;

 Based on this results it is hard to say if KRPBE is safe for hypertensive patients and the effectiveness of this extract must be confirmed in clinical trials.

The sentence: “Our study suggests that KRPBE rich in(….) “ page 10, lines 370-371 was revised accordingly (lines 364-366). Like Pycnogenl (French maritime pine bark extract), KRPBE is available in dietary supplements and multi-vitamins in South Korea. We think that it is safe.