Anti-Estrogenic Activity of Guajadial Fraction, from Guava Leaves (Psidium guajava L.)

Round 1

Reviewer 1 Report

Comments

Present manuscript summarises results of nicely designed in vitro and in vivo studies focussed on anticarcinogenic activity of  Guajadial, a caryophyllene-based meroterpenoid, which was shown to be the major constituent of dichloromethane extract (DCE) from the crude P. guajava leaves. Purified fraction “Final” showed promising antiproliferative activity against several human cancer cell lines. For the first time, authors showed that the enriched guajadial fraction had also anti-estrogenic activity in vivo and inhibited proliferative effect of estradiol on the uterus of pre-pubescent rats. These results suggested that anticancer effect of guajadial enriched fraction does not involve exclusively the action on estrogenic receptors, but also cell death mechanism activation. Experiments were performed precisely and results are clearly described. English language is good. 

 

Could authors provide explanations of following results:

 

1. Results: 3.2.3. Toxicity of Final fractions was observed for the Ehrlich solid tumor test and as body weight variations, which were the most prominent for dose of 12,5 mg/kg-1 in the Hollow fiber experiment in mice showing highest body weight loss. By contrast, this lowest dose (12.5 mg.kg-1) was shown to be the most efficient on inhibition of cell growth of human MCF-7 in vivo.
2. Results: 3.2.4. Authors showed that the treatment with “Final” fraction after 15 minutes of estradiol (E2) administration was able to significantly inhibit the proliferative effect of treatment on uterus but  did not alter the relative weight of the ovaries of prepubertal rats. Could you comment on this difference?

Author Response

Reviewer #1:

                  Present manuscript summarises results of nicely designed in vitro and in vivo studies focussed on anticarcinogenic activity of Guajadial, a caryophyllene-based meroterpenoid, which was shown to be the major constituent of dichloromethane extract (DCE) from the crude P. guajava leaves. Purified fraction “Final” showed promising antiproliferative activity against several human cancer cell lines. For the first time, authors showed that the enriched guajadial fraction had also anti-estrogenic activity in vivo and inhibited proliferative effect of estradiol on the uterus of pre-pubescent rats. These results suggested that anticancer effect of guajadial enriched fraction does not involve exclusively the action on estrogenic receptors, but also cell death mechanism activation. Experiments were performed precisely and results are clearly described. English language is good. 

            Could authors provide explanations of following results:

 

1. Results: 3.2.3. Toxicity of Final fractions was observed for the Ehrlich solid tumor test and as body weight variations, which were the most prominent for dose of 12,5 mg/kg-1 in the Hollow fiber experiment in mice showing highest body weight loss. By contrast, this lowest dose (12.5 mg.kg-1) was shown to be the most efficient on inhibition of cell growth of human MCF-7 in vivo.
   

It is not possible to confirm that 12.5 mg.kg-1 dose was toxic because its body weight variation was non-significant. (Figure 10)

 

1. Results: 3.2.4. Authors showed that the treatment with “Final” fraction after 15 minutes of estradiol (E2) administration was able to significantly inhibit the proliferative effect of treatment on uterus but did not alter the relative weight of the ovaries of prepubertal rats. Could you comment on this difference?

 

The treatment with FFINAL after 15 minutes of estradiol (E2) administration was able to significantly inhibit proliferative effect on uterus, however, did not alter the relative weight of the prepubertal rats ovaries. This fact can be explained through the hormones involved in this process. Pituitary hormones FSH (follicle stimulating hormone) and LH (luteinizing hormone) are related to ovarian stimulation and menstrual cycle. Hypothalamus regulates the release of pituitary hormones and is regulated by estrogen and progesterone (produced by the ovaries). Elevated levels of estrogens and progesterone stop the pituitary gland stimulation and therefore, blocking estrous cycle and consequently inhibiting ovulation. In prepubertal animals, this cycle is not yet active and, therefore, the ovary is not producing estrogens, which would influence the test results. The ovaries are mainly influenced by chorionic gonadotropin (produced by placenta), FSH and LH during prepubertal phase. These hormones when injected into the prepubertal rat will produce ovarian stimulation characterized by increased blood irrigation. In the test used, there would be changes in the ovaries only if the guava's active ingredients had an effect on the FSH and LH ovarian receptors (discussed in Section 4).

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript No molecules-732503 entitled “Antiestrogenic activity of guajadial fraction, from  Guava Leaves (Psidium guajava L.)” by Bazioli et al may be published in Molecules after major revision.                                                                                                                                       Specific comments                                                                                                                                  The Authors described the study about evaluation of the anti-estrogenic action and antiproliferative activity of guajadial . Therefore, the enriched guajadial fraction from the crude P. guajava was checked in terms of antiproliferative activity in vitro. The results confirmed its selectivity for human breast cancer cell  lines MCF-7 and MCF-7 BUS. Furthermore, anti-estrogenic activity in vivo was also tested. The obtained data indicated that guajadial enriched fraction inhibited the proliferative effect of estradiol on the uterus of pre-pubescent rats. The antiproliferative and anti-estrogenic activity of guajadial is probably corelated to structural similarity to tamoxifen which also causes tumor inhibition through estrogen receptors. The paper may be interesting for the readers but there are many points which should be corrected.   1. 2. Material and Methods. There are no data describing producers of many reagents used in the study inluding anisaldehyde, hexane, dichloromethane, dicloromethane?, methanol, acetic acid, p-anisaldehyde, sulfuric acid, DMSO, doxorubicin, colchicine. It should be corrected. 2. 2.3. Extraction and fractionation . The Authors decribed that” Elutent polarity was increased by gradients of hexane, dicloromethane and methanol. Obtained fractions were grouped according to thin-layer chromatography (TLC) profile [visualized with anisaldehyde reagent (acetic acid, p-anisaldehyde, sulfuric acid (48:1:1)) followed by heating at 110°C.], resulting in three fractions named A, B and C. Fraction B (14 g) was further fractionated by flash column chromatography over Silica gel 60 (Merck 0,04 - 0,063 mm thickness) with an isocratic system of dicloromethane and 0,3 % methanol. Five fractions were obtained based on TLC profile and named B1, B2, B3, B4, B5. B2 fraction (1.6 g) was fractionated on reverse phase C-18 column (Phenomenex, 55 M, 70 A, 5 g/20 mL) with eluting mobile phase of deionized water, acetonitrile/water mixture (95:5, v/v) and methanol, resulting in five fractions. One of the resulting fractions was a solid (600 mg) fraction  named FFINAL, containing active compounds. FFINAL was tested by in vitro and in vivo assays and chemically characterized by GC-MS.” a)      The graphical presentation of TLC results showing fractions A, B and C should be presented in Supplementary material. b)     Why did the Authors select fraction B to further chromatographic study? It should be explained in the manuscript. c)      The graphical presentation of TLC results showing fractions B1-B5 should be presented in Supplementary material. d)     Why did the Authors select fraction B2 to further chromatographic study? It should be explained in the manuscript. e)      The detailed description of time collection for all fractions should be clearly reported in the manuscript. Which of them was FFinal. The chromatogram of this analysis should be included in supplementary material. f)       Why did the Authors select this fraction (FFinal) to further study? It should be explained in the manuscript.   3. 2.6. In vivo assays.2.6.1. Animals How many mice and rats were used during all experiments reported in the paper? It should be clearly reported in the manuscript. 4. 2.9.2. Statistical analysis. The Authors used one-way or two-way ANOVA followed by Tukey’s post-hoc test for the statistical evaluation of the obtained results. ANOVA test belongs to parametric test and it may be used only in situation when a normal distribution of variables was confirmed. There are no information whether it was checked. If it was, which test was used. It should be reported in the manuscript. 5. Figures 1-10. How many experiments were performed in each study. It should be clearly reported in the manuscript. Minor editorial mistake In the whole manuscript there are a lot of editorial mistakes such as: Abstract: TGI = 5,59 1. Introduction: line 36-  The references [3,4] should be omitted. 2.3. Extraction and fractionation: (Merck 0,0063- 0,200 mm ; (Merck 0,04- 0,063 mm 2.5.1. Cell lines and culture conditions:  (Nutricell, 1000 U/ml:1000 g/ml)] Table 1 : D.I.h  (104 cel ml-1) 2.5.2. Sample dilution:  μl; 1000 U/ml:1000 g/ml)]; Doxorubicine; 0.25nM;  2.5.3. Antiproliferative assay . μg.ml-1 2.5.4. E-screen assay. 72h; μg ml-1; μg/mL; 144h. 2.5.5. Cell cycle analysis: ml-1; 24h; 48h; 1 mL 140 ml-1); 24h;μg ml-1; 12h; 4 ºC, 200 μl 2.7. Acute oral toxicity: (Anasedan® 30mg/kg, i.p.) 2.8.3. Hollow Fiber assay: (1000 U/ml:1000 g/ml)]; 24h; (0.55 205 mg/ml, 1 ml/well); (250 μl/well); (Anasedan® 30mg/kg, i.p.); CO2; 4h 3.1.1. In vitro antiproliferative screening:  (TGI = 27,23 μg.mL-1, OVCAR-3); (TGI = 39,94 μg.mL-1, PC-3); Table 2. The description of Table 2 should be corrected: (DCE?) and doxorrubicin?  3.1.5. MCF-7 BUS cell cycle by flow cytometry: Colchicine (0,25 μg.mL-1); 48h,  3.2.1. Acute toxicity: FFINAL; 2h;   3.2.2. Ehrlich solid tumor (back): doxorrubicin   These editorial mistakes should be corrected. Summarizing, the manuscript can be publication in Molecules after major revising.

Author Response

Reviewer #2:

                  The Authors described the study about evaluation of the anti-estrogenic action and antiproliferative activity of guajadial. Therefore, the enriched guajadial fraction from the crude P. guajava was checked in terms of antiproliferative activity in vitro. The results confirmed its selectivity for human breast cancer cell lines MCF-7 and MCF-7 BUS. Furthermore, anti-estrogenic activity in vivo was also tested. The obtained data indicated that guajadial enriched fraction inhibited the proliferative effect of estradiol on the uterus of pre-pubescent rats. The antiproliferative and anti-estrogenic activity of guajadial is probably corelated to structural similarity to tamoxifen which also causes tumor inhibition through estrogen receptors. The paper may be interesting for the readers but there are many points which should be corrected.

 

1. 2. Material and Methods. There are no data describing producers of many reagents used in the study inluding anisaldehyde, hexane, dichloromethane, dicloromethane?, methanol, acetic acid, p-anisaldehyde, sulfuric acid, DMSO, doxorubicin, colchicine. It should be corrected.

Section 2.1 Commercial reagents were added in the manuscript.

2. 2.3. Extraction and fractionation. The Authors decribed that” Elutent polarity was increased by gradients of hexane, dicloromethane and methanol. Obtained fractions were grouped according to thin-layer chromatography (TLC) profile [visualized with anisaldehyde reagent (acetic acid, p-anisaldehyde, sulfuric acid (48:1:1)) followed by heating at 110°C.], resulting in three fractions named A, B and C. Fraction B (14 g) was further fractionated by flash column chromatography over Silica gel 60 (Merck 0,04 - 0,063 mm thickness) with an isocratic system of dicloromethane and 0,3 % methanol. Five fractions were obtained based on TLC profile and named B1, B2, B3, B4, B5.

B2 fraction (1.6 g) was fractionated on reverse phase C-18 column (Phenomenex, 55 mM, 70 A, 5 g/20 mL) with eluting mobile phase of deionized water, acetonitrile/water mixture (95:5, v/v) and methanol, resulting in five fractions. One of the resulting fractions was a solid (600 mg) fraction named FFINAL, containing active compounds. FFINAL was tested by in vitro and in vivo assays and chemically characterized by GC-MS.”

1. a)      The graphical presentation of TLC results showing fractions A, B and C should be presented in Supplementary material.

Now, It is represented in Figure S1.

1. b)     Why did the Authors select fraction B to further chromatographic study? It should be explained in the manuscript.

                  Fraction B was selected after GC-MS analysis showing that guajadial was present in this fraction. Now it is explained in section 2.4

1. c)      The graphical presentation of TLC results showing fractions B1-B5 should be presented in Supplementary material.

Now, It is represented in Figure S2.

1. d)     Why did the Authors select fraction B2 to further chromatographic study? It should be explained in the manuscript.

                  Fraction B3 was selected after GC-MS analysis showing that guajadial was present in this fraction. Now it is explained in section 2.4

1. e)      The detailed description of time collection for all fractions should be clearly reported in the manuscript. Which of them was FThe chromatogram of this analysis should be included in supplementary material.

                  The collected volume of each fraction (50, 20 and 2 mL) were added in section 2.4. Fraction B3.2 was named FFINAL and the TLC is represented in Figure S3.

1. f)       Why did the Authors select this fraction (F_Final)to further study? It should be explained in the manuscript.  

Fraction B3.2 (FFINAL) was selected after GC-MS analysis showing that guajadial was present in this fraction. Now it is explained in section 2.4

3. 2.6. In vivo assays.2.6.1. Animals

How many mice and rats were used during all experiments reported in the paper? It should be clearly reported in the manuscript.

                  Now, the number of animals is reported in the manuscript (sections 2.7.1, 2.8, 2.9.1, 2.9.2)

4. 2.9.2. Statistical analysis. The Authors used one-way or two-way ANOVA followed by Tukey’s post-hoc test for the statistical evaluation of the obtained results. ANOVA test belongs to parametric test and it may be used only in situation when a normal distribution of variables was confirmed. There are no information whether it was checked. If it was, which test was used. It should be reported in the manuscript.

                  Statistical analysis was performed/revised by one-way non-parametric ANOVA (Kruskal-Wallis test) (section 2.11).

5. Figures 1-10. How many experiments were performed in each study. It should be clearly reported in the manuscript.

For each study was performed a single experiment with triplicate determinations (section 2.11).

Minor editorial mistake

In the whole manuscript there are a lot of editorial mistakes such as:

Abstract: TGI = 5,59

1. Introduction: line 36- The references [3,4] should be omitted.

2.3. Extraction and fractionation: (Merck 0,0063- 0,200 mm ; (Merck 0,04- 0,063 mm

2.5.1. Cell lines and culture conditions:  (Nutricell, 1000 U/ml:1000 g/ml)]

Table 1 : D.I.h  (104 cel ml-1)

2.5.2. Sample dilution:  μl; 1000 U/ml:1000 g/ml)]; Doxorubicine; 0.25nM;

 2.5.3. Antiproliferative assay . μg.ml^-1

2.5.4. E-screen assay. 72h; μg ml-1; μg/mL; 144h.

2.5.5. Cell cycle analysis: ml-1; 24h; 48h; 1 mL 140 ml-1); 24h;μg ml-1; 12h; 4 ºC, 200 μl

2.7. Acute oral toxicity: (Anasedan® 30mg/kg, i.p.)

2.8.3. Hollow Fiber assay: (1000 U/ml:1000 g/ml)]; 24h; (0.55 205 mg/ml, 1 ml/well); (250 μl/well); (Anasedan® 30mg/kg, i.p.); CO2; 4h

3.1.1. In vitro antiproliferative screening:  (TGI = 27,23 μg.mL^-1, OVCAR-3); (TGI = 39,94 μg.mL^-1, PC-3);

Table 2. The description of Table 2 should be corrected: (DCE?) and doxorrubicin?

 3.1.5. MCF-7 BUS cell cycle by flow cytometry: Colchicine (0,25 μg.mL^-1); 48h,

 3.2.1. Acute toxicity: FFINAL; 2h;  

                  The English and units were also extensively revised in the new version of the manuscript and all the corrections above suggested by the reviewer were made in the manuscript. The authors appreciate the critical corrections.

3.2.2. Ehrlich solid tumor (back): doxorubicin

                  A comparison of F_FINAL with doxorubicin treatment was included.

These editorial mistakes should be corrected.

Summarizing, the manuscript can be publication in Molecules after major revising.

Reviewer 3 Report

Carvalho et al. studied thoroughly the biological activity of guajadial fraction from Guava leaves. Ffinal fraction contained 3 major compounds.

The chromatographic analysis of Ffinal fraction should be the first among the in vitro results (3.1.1 section), because this is the starting point of the work.

The structure of guajadial and its stereoisomer should be given in the manuscript.

The fragmentation profiles all of three major compounds should be given in the supporting material.

The following errors should be corrected: 1. page 21. line: sequential,

5. page 212. line: doxorubicin; Table 2.: doxorubicin.

The manuscript is suitable for publication with minor revision.

Author Response

Reviewer #3:

                  Carvalho et al. studied thoroughly the biological activity of guajadial fraction from Guava leaves. Ffinal fraction contained 3 major compounds.

The chromatographic analysis of Ffinal fraction should be the first among the in vitro results (3.1.1 section), because this is the starting point of the work.

                  Now, 3.1 Chromatographic analysis of F_FINAL is the first topic of the result section.

The structure of guajadial and its stereoisomer should be given in the manuscript.

                  Guajadial and its epimer’s structures are represented in Figure 1 (introduction).

The fragmentation profiles all of three major compounds should be given in the supporting material.

Tandem spectrum of compounds at RT 45.01 and 46.71 min are represented in Figures S4B and S4C. Since they are epimers, the fragmentation spectrums are very similar. The compound at RT 28.08 min is due phthalate contamination in the solvent used to prepare the sample for GC analysis.  Since this compound are not present in FFINAL fraction, we did not give the tandem spectrum. Now, these informations are reported in section 3.1.

The following errors should be corrected: 1. page 21. line: sequential,

1. page 212. line: doxorubicin; Table 2.: doxorubicin.

The English and units were also extensively revised in the new version of the manuscript and all the corrections above suggested by the reviewer were made in the manuscript. The authors appreciate the critical corrections.

The manuscript is suitable for publication with minor revision.

Round 2

Reviewer 2 Report

The revised manuscript No molecules-732503 entitled “Antiestrogenic activity of guajadial fraction, from  Guava Leaves (Psidium guajava L.)” by  Bazioli et al in this form can  may be published in Molecules.