Spondias mombin Seed Oil Compounds Identification by Raman Spectroscopy and NMR

Round 1

Reviewer 1 Report

Dear Authors, 

thank you for this interesting insight into essential oil (EO) of Spondias mombin seeds. 

The manuscript describes analysis of the EO with the use of RAMAN an MMR, as well as cancer cells. Recently, many authors focused on studying the essential oils in various aspects from their chemical analysis to potential medicinal properties.

Several times you underline that your Raman analysis is the first on this particular EO. However, please note, that it does not necessarily prove novelty. Multiple studies have been conducted on EOs that have used Raman technique.

Before the manuscript can be recommended for publication, please consider following remarks.

Introduction: This part is very scarce. You should do better in introducing the topic. Please stress more on the novelty. Furthermore, please state the aim of your research at the end of this section. 

Material and methods: Here, I would like to focus on the studies that you have conducted on cancer cells. Please comment on the controls. Have you used any solvent-only control? It should be included in the results. 

Results and Discussion:

In this part, my main issue is the connection that you claim between antioxidative properties and viability. I think that the used methodology is not enough to prove such a correlation. Have you considered that your cells could simply use the organic matter from the extract and build more biomass? This part is not acceptable in the present form. You should either clearly state this issue or add more studies to prove your claims. You should also discuss the results regarding cells' physiology. Otherwise, it's just speculation. 

Moreover, please add some references in the first sentence of this paragraph.

Conclusions: You don't even mention the studies on cancer cells in this part and you should if they were included in the research. 

References: I understand that older papers are also great, but I feel that you should use more than ca. 11 papers that are no older than 5 years. This would allow you to better show the novelty. 

I have found some minor English errors (such as missing commas or articles) in the text. Please check it again. 

I hope that my comments will be helpful in improving the manuscript. 

I wish you luck in the revision and hope to see the revised manuscript. 

Author Response

Comment 1:

Several times you underline that your Raman analysis is the first on this particular EO. However, please note, that it does not necessarily prove novelty. Multiple studies have been conducted on EOs that have used Raman technique.

 

Answer: Your observation is correct, we have removed the phrase "for the first time", and included more support references for the introduction section. In that part what we wanted to express is about the analysis of the oil extracted specifically from the seed of Spondias mombin.

 

 

Comment 2:

Introduction: This part is very scarce. You should do better in introducing the topic. Please stress more on the novelty. Furthermore, please state the aim of your research at the end of this section.

 

Answer: The introduction section was updated and 10 references were added that support some paragraphs (Ref 10-24).

10. Di Pietro, M. E., Mannu, A., & Mele, A. NMR Determination of Free Fatty Acids in Vegetable Oils. Processes. 2020, 8(4), 410. https://doi.org/10.3390/pr8040410
11. Huck, C. W., Bec, K. B., & Grabska, J. The use of vibrational spectroscopy in medicinal plant analysis: current and future directions. Planta Medica. 2019, 85(18), 1408-1409. 10.1055/s-0039-3399687
12. Krafft, C., & Popp, J. Micro-Raman spectroscopy in medicine. Physical Sciences Reviews. 2019, 4(10), 1-15. https://doi.org/10.1515/psr-2017-0047
13. De Silva, I. W., Kretsch, A. R., Lewis, H. M., Bailey, M., & Verbeck, G. F. True one cell chemical analysis: a review. Analyst. 2019, 144(16), 4733-4749. https://doi.org/10.1039/C9AN00558G
14. Nims, C., Cron, B., Wetherington, M., Macalady, J., & Cosmidis, J. Low frequency Raman spectroscopy for micron-scale and in vivo characterization of elemental sulfur in microbial samples. Scientific reports. 2019, 9(1), 1-12. https://doi.org/10.1038/s41598-019-44353-6
15. He, H., Sun, D. W., Pu, H., Chen, L., & Lin, L. Applications of Raman spectroscopic techniques for quality and safety evaluation of milk: A review of recent developments. Critical reviews in food science and nutrition. 2019, 59(5), 770-793. https://doi.org/10.1080/10408398.2018.1528436
16. Hu, R., He, T., Zhang, Z., Yang, Y., & Liu, M. Safety analysis of edible oil products via Raman spectroscopy. Talanta. 2019, 191, 324-332. https://doi.org/10.1016/j.talanta.2018.08.074
17. Meenu, M., Cai, Q., & Xu, B. A critical review on analytical techniques to detect adulteration of extra virgin olive oil. Trends in Food Science & Technology. 2019, 91, 391-408. https://doi.org/10.1016/j.tifs.2019.07.045
18. Berghian-Grosan, C., & Magdas, D. A.Raman spectroscopy and Machine-Learning for edible oils evaluation. Talanta. 2020, 121176. https://doi.org/10.1016/j.talanta.2020.121176
19. Kwofie, F., Lavine, B. K., Ottaway, J., & Booksh, K. Incorporating brand variability into classification of edible oils by Raman spectroscopy. Journal of Chemometrics. 2020, 34(7), e3173. https://doi.org/10.1002/cem.3173
20. Saraiva, A. G. Q., Saraiva, G. D., Albuquerque, R. L., Nogueira, C. E. S., Teixeira, A. M. R., Lima, L. B., ... & de Sousa, F. F. Chemical analysis and vibrational spectroscopy study of essential oils from Lippia sidoides and of its major constituent. Vibrational Spectroscopy. 2020, 103111. https://doi.org/10.1016/j.vibspec.2020.103111
21. Pedro, A. C., Bach, F., Stafussa, A. P., Menezes, L. R. A., Barison, A., Maciel, G. M., & Haminiuk, C. W. I. 1H NMR and Raman spectroscopy of oils and extracts obtained from organic and conventional goji berries: yield, fatty acids, carotenoids and biological activities. International Journal of Food Science & Technology. 2019, 54(1), 282-290. https://doi.org/10.1111/ijfs.13976
22. Ferreira, B. S., de Almeida, C. G., Le Hyaric, M., de Oliveira, V. E., Edwards, H. G., & de Oliveira, L. F. C. Raman spectroscopic investigation of carotenoids in oils from Amazonian products. Spectroscopy Letters. 2013, 46(2), 122-127. https://doi.org/10.1080/00387010.2012.693569
23. Gamsjaeger, S., Baranska, M., Schulz, H., Heiselmayer, P., & Musso, M. Discrimination of carotenoid and flavonoid content in petals of pansy cultivars (Viola x wittrockiana) by FT‐Raman spectroscopy. Journal of Raman Spectroscopy. 2011, 42(6), 1240-1247. https://doi.org/10.1002/jrs.2860
24. Bhosale, P., Ermakov, I. V., Ermakova, M. R., Gellermann, W., & Bernstein, P. S. Resonance Raman quantification of nutritionally important carotenoids in fruits, vegetables, and their juices in comparison to high-pressure liquid chromatography analysis. Journal of Agricultural and Food Chemistry. 2004, 52(11), 3281-3285. https://doi.org/10.1021/jf035345q

 

Comment 3:

Material and methods: Here, I would like to focus on the studies that you have conducted on cancer cells. Please comment on the controls. Have you used any solvent-only control? It should be included in the results.

 

Answer: In the Samples preparation and cell culture section, the description of this section was updated and it was supported by three references previously reported.

 

A stock solution (100 mg mL-1) of Spondias mombin L. seed oil solubilized with ethyl alcohol (1:4) was prepared. Prior to the cell experiments, these samples were diluted to final concentrations of 1, 10, 50, 100, and 500 μg/mL using culture medium. HTC 116 cells (obtained from American Type Culture Collection: ATCC) were grown in 100 mm cell culture dish at 37 °C with 5% CO2 in DMEM medium, supplemented with 10% fetal bovine serum; the cells were placed on 96 well cell culture plate for cell proliferation assay. The oil is free of any solvent except from alcohol used for the final dilution, in a concentration range from 0.00075% to 0.375%, these concentrations do not interfere with the assay. Standard protocols contain ≈ 2% alcohol concentration as previously reported [26-27]. Effects on cell viability by ethyl alcohol have been reported at a concentration of 5% and above [28].

 

26. Döll-Boscardin, P. M., Sartoratto, A., Sales Maia, B. H. L. D. N., Padilha de Paula, J., Nakashima, T., Farago, P. V., & Kanunfre, C. C. In vitro cytotoxic potential of essential oils of Eucalyptus benthamii and its related terpenes on tumor cell lines. Evidence-Based Complementary and Alternative Medicine. 2012, 342652, 1-8. https://doi.org/10.1155/2012/342652
27. Virador, V. M., Kobayashi, N., Matsunaga, J., & Hearing, V. J. A standardized protocol for assessing regulators of pigmentation. Analytical biochemistry. 1999, 270(2), 207-219. 10.1006/abio.1999.4090
28. Forman, S., Káˇs, J., Fini, F., Steinberg, M., & Ruml, T. The effect of different solvents on the ATP/ADP content and growth properties of HeLa cells. Journal of biochemical and molecular toxicology. 1999, 13(1), 11-15. https://doi.org/10.1002/(SICI)1099-0461(1999)13:1<11::AID-JBT2>3.0.CO;2-R

 

Comment 4:

In this part, my main issue is the connection that you claim between antioxidative properties and viability. I think that the used methodology is not enough to prove such a correlation. Have you considered that your cells could simply use the organic matter from the extract and build more biomass? This part is not acceptable in the present form. You should either clearly state this issue or add more studies to prove your claims. You should also discuss the results regarding cells’ physiology. Otherwise, it’s just speculation. Moreover, please add some references in the first sentence of this paragraph.

 

Answer: The observations described in the previous paragraph were addressed and supported with some references.

 

55. Pucci, B., Kasten, M., & Giordano, A. Cell cycle and apoptosis. Neoplasia. 2000, 2(4), 291-299. https://doi.org/10.1038/sj.neo.7900101
56. Ali, S. S., Ahsan, H., Zia, M. K., Siddiqui, T., & Khan, F. H. Understanding oxidants and antioxidants: Classical team with new players. Journal of Food Biochemistry. 2020, 44(3), e13145. https://doi.org/10.1111/jfbc.13145
57. Ruhee, R. T., Ma, S., & Suzuki, K. Protective effects of sulforaphane on exercise-induced organ damage via inducing antioxidant defense responses. Antioxidants. 2020, 9(2), 136. https://doi.org/10.3390/antiox9020136

 

Comment 5:

Conclusions: You don't even mention the studies on cancer cells in this part and you should if they were included in the research.

 

Answer: The conclusion section was also updated.

 

In the present work, we demonstrate the use of Raman and ¹H- and ¹³C-NMR spectroscopy as non-invasive and non-destructive analytical techniques, which allow characterizing the oil extracted from the seed of Spondias mombin L. Through the use of these two techniques, the characteristic vibrational frequencies of the Spondias mombin L. seed oil were identified, as well as its fatty acid profile. Raman vibratory frequencies at 1006, 1158 and 1523 cm^-1 were identified and assigned to carotenoid compounds with antioxidant activity. On the other hand, NMR experiments proved to be a valuable tool to obtain the fatty acid profile of crude oil from the seed. Selected peak integrals were used to calibrate the composition of MUFA, PUFA and SFA. Applying this approach to a Spondias mombin L. seed oil sample, we obtained values for MUFA and PUFA contents of 29.4 and 43.5%, which indicated that the oil is highly unsaturated (> 70%). The oil showed high biocompatibility and promotes cell viability in the HTC 116 cell line.

Due to its properties, the seed oil of this tropical fruit could have a potential application in cosmetic and pharmaceutical products as an antioxidant agent. Finally, the combination of Raman spectroscopy and the NMR technique are very powerful and useful tools for the characterization of organic compounds, particularly in natural vegetable oils.

 

Comment 6:

References: I understand that older papers are also great, but I feel that you should use more than ca. 11 papers that are no older than 5 years. This would allow you to better show the novelty.

Answer: Some sections of the manuscript were updated and it was supported with more recent references. English writing was also reviewed in all sections.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments:

This study deals with the analyse of Spondias mombin seed oil by using Raman spectroscopy and NMR. The identification of S. mombin oil is not novel, which was also mentioned in the introduction section. The interesting part of this manuscript is the Raman spectroscopy and the combination with S. mombin oil properties (e.g. antioxidant). Is it possible to connect the data from the Raman spectroscopy with the results from the antioxidant activity (e.g. PLS method)? The manuscript cannot accept for the Applied Sciences – Section Optics and Lasers without changes. Below the authors will find some comments:

Introduction

Please rework this section. The authors have to describe the results from previous studies and not only mentioned the used methods.

What are the results of other Raman spectroscopy studies for carotenoids? For example, Gamsjaeger, Baranska, Schulz, Heiselmayer, Musso (2010) Discrimination of carotenoid and flavonoid content in petals of pansy cultivars by FT-Raman spectroscopy. Journal of Raman Spectroscopy 42:1240-1247;

Results and Discussion

Are carotenoids the main component in the S. mombin seed oil? What does the results from other studies show?

Page 4: line 156: Figure 2. Region of the Raman spectrum where Raman frequencies are located … associated with carotenoids.

Which samples did you use (e.g. S. mombin, or carotenoids)?

Page 5: line 175: CH2 and CH3 are Raman frequencies commonly found in vegetable oil spectra; these are common structures in oils; therefore, the identification of the compound is unclear [14, 17]. What does it mean? Please check also Conclusion section. Line 296: This fatty acid profile of the seed crude oil was characterized using different analytic techniques such as Raman, … Is the Raman spectroscopy useful for analysing oils?

Conclusions

Please do not only mention what have you done? For example, line 296: This fatty acid profile of the seed crude oil was characterized using different analytic techniques such as Raman, 1H- and 13C-NMR.

Author Response

Comment 1:

Introduction

Please rework this section. The authors have to describe the results from previous studies and not only mentioned the used methods. What are the results of other Raman spectroscopy studies for carotenoids? For example, Gamsjaeger, Baranska, Schulz, Heiselmayer, Musso (2010) Discrimination of carotenoid and flavonoid content in petals of pansy cultivars by FT-Raman spectroscopy. Journal of Raman Spectroscopy 42: 1240-1247;

 

Answer: he introduction section has been updated and references that support the modifications have been included (Ref 10 to 24).

 

10. Di Pietro, M. E., Mannu, A., & Mele, A. NMR Determination of Free Fatty Acids in Vegetable Oils. Processes. 2020, 8(4), 410. https://doi.org/10.3390/pr8040410
11. Huck, C. W., Bec, K. B., & Grabska, J. The use of vibrational spectroscopy in medicinal plant analysis: current and future directions. Planta Medica. 2019, 85(18), 1408-1409. 10.1055/s-0039-3399687
12. Krafft, C., & Popp, J. Micro-Raman spectroscopy in medicine. Physical Sciences Reviews. 2019, 4(10), 1-15. https://doi.org/10.1515/psr-2017-0047
13. De Silva, I. W., Kretsch, A. R., Lewis, H. M., Bailey, M., & Verbeck, G. F. True one cell chemical analysis: a review. Analyst. 2019, 144(16), 4733-4749. https://doi.org/10.1039/C9AN00558G
14. Nims, C., Cron, B., Wetherington, M., Macalady, J., & Cosmidis, J. Low frequency Raman spectroscopy for micron-scale and in vivo characterization of elemental sulfur in microbial samples. Scientific reports. 2019, 9(1), 1-12. https://doi.org/10.1038/s41598-019-44353-6
15. He, H., Sun, D. W., Pu, H., Chen, L., & Lin, L. Applications of Raman spectroscopic techniques for quality and safety evaluation of milk: A review of recent developments. Critical reviews in food science and nutrition. 2019, 59(5), 770-793. https://doi.org/10.1080/10408398.2018.1528436
16. Hu, R., He, T., Zhang, Z., Yang, Y., & Liu, M. Safety analysis of edible oil products via Raman spectroscopy. Talanta. 2019, 191, 324-332. https://doi.org/10.1016/j.talanta.2018.08.074
17. Meenu, M., Cai, Q., & Xu, B. A critical review on analytical techniques to detect adulteration of extra virgin olive oil. Trends in Food Science & Technology. 2019, 91, 391-408. https://doi.org/10.1016/j.tifs.2019.07.045
18. Berghian-Grosan, C., & Magdas, D. A.Raman spectroscopy and Machine-Learning for edible oils evaluation. Talanta. 2020, 121176. https://doi.org/10.1016/j.talanta.2020.121176
19. Kwofie, F., Lavine, B. K., Ottaway, J., & Booksh, K. Incorporating brand variability into classification of edible oils by Raman spectroscopy. Journal of Chemometrics. 2020, 34(7), e3173. https://doi.org/10.1002/cem.3173
20. Saraiva, A. G. Q., Saraiva, G. D., Albuquerque, R. L., Nogueira, C. E. S., Teixeira, A. M. R., Lima, L. B., ... & de Sousa, F. F. Chemical analysis and vibrational spectroscopy study of essential oils from Lippia sidoides and of its major constituent. Vibrational Spectroscopy. 2020, 103111. https://doi.org/10.1016/j.vibspec.2020.103111
21. Pedro, A. C., Bach, F., Stafussa, A. P., Menezes, L. R. A., Barison, A., Maciel, G. M., & Haminiuk, C. W. I. 1H NMR and Raman spectroscopy of oils and extracts obtained from organic and conventional goji berries: yield, fatty acids, carotenoids and biological activities. International Journal of Food Science & Technology. 2019, 54(1), 282-290. https://doi.org/10.1111/ijfs.13976
22. Ferreira, B. S., de Almeida, C. G., Le Hyaric, M., de Oliveira, V. E., Edwards, H. G., & de Oliveira, L. F. C. Raman spectroscopic investigation of carotenoids in oils from Amazonian products. Spectroscopy Letters. 2013, 46(2), 122-127. https://doi.org/10.1080/00387010.2012.693569
23. Gamsjaeger, S., Baranska, M., Schulz, H., Heiselmayer, P., & Musso, M. Discrimination of carotenoid and flavonoid content in petals of pansy cultivars (Viola x wittrockiana) by FT‐Raman spectroscopy. Journal of Raman Spectroscopy. 2011, 42(6), 1240-1247. https://doi.org/10.1002/jrs.2860
24. Bhosale, P., Ermakov, I. V., Ermakova, M. R., Gellermann, W., & Bernstein, P. S. Resonance Raman quantification of nutritionally important carotenoids in fruits, vegetables, and their juices in comparison to high-pressure liquid chromatography analysis. Journal of Agricultural and Food Chemistry. 2004, 52(11), 3281-3285. https://doi.org/10.1021/jf035345q

 

 

Comment 2:

Results and Discussion

Are carotenoids the main component in the S. mombin seed oil? What does the results from other studies show? Page 4: line 156: Figure 2. Region of the Raman spectrum where Raman frequencies are located … associated with carotenoids. Which samples did you use (e.g. S. mombin, or carotenoids)?.

 

Answer: The results and discussions section was updated, as well as the title of figure 2 was supplemented.

 

Comment 3:

Page 5: line 175: CH2 and CH3 are Raman frequencies commonly found in vegetable oil spectra; these are common structures in oils; therefore, the identification of the compound is unclear [14, 17]. What does it mean? Please check also Conclusion section. Line 296: This fatty acid profile of the seed crude oil was characterized using different analytic techniques such as Raman, … Is the Raman spectroscopy useful for analysing oils?

 

Answer: The items related to all the observations mentioned were updated and modified.

 

Raman spectra recorded from Spondias mombin L. vegetable oil samples show signals associated with the vibrational modes C-C (ν), C-H (δ), C= O (ν), C=C (ν), as well as bands assigned to functional groups such as CH₂ and CH₃ [32, 35]

 

Comment 4:

 Conclusions

Please do not only mention what have you done? For example, line 296: This fatty acid profile of the seed crude oil was characterized using different analytic techniques such as Raman, 1H- and 13C-NMR.

 

Answer: The conclusions section was updated.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors, 

thank you for the revision.  

However, I have some additional remarks that you should consider before the paper is published. 

Introduction: The last sentence could be composed somewhere in the text. The paragraph should end with the clearly stated aim (that is present at the end of the previous paragraph). 

Results and discussion: 

Unfortunately, you have left this problem unsolved. The references are not enough to back it up. How do you know whether your cells were not feeding on the extract? I don't exclude the beneficial value of antioxidants, but please note that it was not the only factor that could lead to the described increase in viability! I still think that you should discuss it thoroughly in the text. The added fragment is in my opinion not enough and oversimplified. 

Best of luck. 

 

 

Author Response

Comment 1:

Introduction

Introduction: The last sentence could be composed somewhere in the text. The paragraph should end with the clearly stated aim (that is present at the end of the previous paragraph).

 

Answer: The last paragraph was adapted in the part of the introduction.

 

“This work is intended to contribute in the development of feasible and accurate methods to characterize natural products composition, and relate them with biological activities and biocompatibility. We performed RS and NMR (1H- and 13C-) as non-invasive and non-destructive analytical techniques to identify molecules profiles through characterization of molecular vibrations and resonant frequencies associated with carotenoids and fatty acid profile of Spondias mombin L. seed oil.”

 

Comment 2:

 

Results and discussion: Unfortunately, you have left this problem unsolved. The references are not enough to back it up. How do you know whether your cells were not feeding on the extract? I don't exclude the beneficial value of antioxidants, but please note that it was not the only factor that could lead to the described increase in viability! I still think that you should discuss it thoroughly in the text. The added fragment is in my opinion not enough and oversimplified.

 

Answer: We have supported this section with some bibliographic references, which I list below.

 

58. Jayathilake, A. G., Senior, P. V., & Su, X. Q. Krill oil extract suppresses cell growth and induces apoptosis of human colorectal cancer cells. BMC complementary and alternative medicine. 2016, 16(1), 328. https://doi.org/10.1186/s12906-016-1311-x
59. Jayathilake, A. G., Kadife, E., Luwor, R. B., Nurgali, K., & Su, X. Q. Krill oil extract suppresses the proliferation of colorectal cancer cells through activation of caspase 3/9. Nutrition & Metabolism. 2019, 16(1), 1-15. https://doi.org/10.1186/s12986-019-0382-3
60. De Stefanis, D., Scimè, S., Accomazzo, S., Catti, A., Occhipinti, A., Bertea, C. M., & Costelli, P. Anti-proliferative effects of an extra-virgin olive oil extract enriched in ligstroside aglycone and oleocanthal on human liver cancer cell lines. Cancers. 2019, 11(11), 1640. https://doi.org/10.3390/cancers11111640Huck, C. W., Bec, K. B., & Grabska, J. The use of vibrational spectroscopy in medicinal plant analysis: current and future directions. Planta Medica. 2019, 85(18), 1408-1409. 10.1055/s-0039-3399687

 

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Dear Authors, 

thank you for the effort in improving the manuscript.

In the present form, I find it suitable for publication. Therefore, I am happy to recommend the article for publication.

Best of luck in your future endeavours.