Neuroprotective and Antiherpetic Properties of Polyphenolic Compounds from Maackia amurensis Heartwood

review’s comments

our answer

1. The results are well-detailed, but I think the authors should provide more details on how these compounds are correlated to different activities and their exact mechanism of action is not detailed enough.

For the neuroprotective activity, we studied two mechanisms: ROS formation and the effect of polyphenolic compounds on mitochondrial membrane potential. We showed that these mechanisms can be involved in the neuroprotective activity of the studied polyphenolic compounds. These data are presented in the manuscript.

For the antiviral activity, we are planning to carry out a number of experiments to study the effect of polyphenolic compounds from M. amurensis heartwood on different stages of viral replication in Vero cells. Besides, we will increase the number of polyphenolic compounds under study in order to make conclusion on the structure-activity relationship. The results will be published in our future papers.   

2. Moreover, in the abstract, the authors should mention the techniques that were followed to isolate phenolic compounds.

We have made the necessary corrections in Abstract.

3. My other comments are:

Lines 18 and 668, is it “slilbens” or “stilbenes”? (1)

Line 19, add “and” before maackolin (2)

Line 21, One should define “CPE” first (3)

Line 40, missing dot (.) after “stilbenes [1]” (4)

Lines 43, 45, and throughout the whole manuscript, remove space between the two reference numbers. (5)

Line 68, remove dot (.) and add a comma between 21 and 21 (6)

Line 309, why quercetin and ascorbic acid were used as reference compounds? An explanation should be provided. (7)

Line 310, remove µM after 2.0 (8)

We have made the necessary corrections (1-8) throughout the text of the manuscript.

Quercetin and ascorbic acid arte often used as reference compounds to evaluate DPPH scavenging effect and FRAP of various natural polyphenolic compounds [1. Katrin Sak. Dependence of DPPH Radical Scavenging Activity of Dietary Flavonoid Quercetin on Reaction Environment // Mini Rev Med Chem. Vol. 14 (60), 2014; P.: 494 – 504. DOI: 10.2174/1389557514666140622204037;  2.

Sridevi Chigurupati , Sohrab Akhtar Shaikh , Jahidul Islam Mohammad, Kesavanarayanan Krishnan Selvarajan , Appala Raju Nemala, Chu How Khaw, Chun Foo Teoh , Ting Hei Kee. In vitro antioxidant and in vivo antidepressant activity of green synthesized azomethine derivatives of cinnamaldehyde // Indian J Pharmacol. Vol. 49 (3), 2017; P.: 229 – 235 DOI: 10.4103/ijp.IJP_293_16 ]

Reviewer 2

1. Title. Why are only two properties of isolated polyphenolic compounds included in the Title? In addition, the Title does not reflect the part of the work devoted to the establishment of the structure of compound 1.

We suppose that the title of the manuscript should not be changed, because:

1. The main purpose of our work was to study the neuroprotective and antiherpetic activities of polyphenolic compounds from Maackia amurensis heartwood. The neuroprotective properties of polyphenolics are mainly due to their antioxidant activity.

2. It is not necessary to include the structure establishment of 1 into the title, because it is obvious that the structures of polyphenolics must be established before their biotesting.

2. In my opinion, it would be informative to give the HPLC spectra of the total extract and fraction 12 and 14, from which the polyphenolic compounds were isolated. 

We gave the HPLC profiles of fractions 12 and 14 in Suppplementary data (S87, S88), from which the polyphenolic compounds were isolated. The HPLC profile of the total extract from M. amurensis was published previously [Fedoreev, S.A.; Kulesh, N.I.; Glebko, L.I.; Pokushalova, T.V.; Veselova, M.V.; Saratikov, A.S.; Vengerovskii, A.I.; Chuchalin, V.S. Maksar: a preparation based on Amur Maackia. Pharm. Chem. J. 2004, 38, 605–610. DOI: 10.1007/s11094-005-0039-6].

Minor concerns

1.       Please check throughout the text: sTilbene (lines 18, 80, 668)

2.       Figure 1 and line 85: “1a, 1b”  should be replaced by “1 (compound 1)” or “1 (mixture of conformers 1a and 1b)”

We made the necessary corrections (1 and 2) throughout the text.

Reviewer 3

I would have reduced the integral curve in the proton spectrum so that the entire curve is visible and does not protrude out of the drawing area.

We have corrected the ¹H NMR spectra of 1 in Supplementary data according to the review’s comments.

I would also have labelled all peaks in the 13C NMR spectrum (e.g. peaks at about 119 ppm).

It is difficult to label all peaks in the area at 119.5, because their chemical shift values are very close to each other. However, it did not affect the structure elucidation process of compound 1.

Even as the spectra are presented in such detail, it is very difficult to check them. Also because it is unfortunately a mixture of 1a and 1b. Most of the assignments seem plausible and comprehensible. There is one small thing the authors should please check:  The signal at 4.16 ppm for proton H3 (cmp. 1a), described as dd (11.6/3.5) looks in the ¹H and in the HSQC more like a td with a large coupling constant (ca 11 Hz) and a small one (ca 2 Hz).

The same is true for signal at 4.36 ppm (H3 cmp. 1b). I am also a little bit surprised that the 13C signal at 38.4 ppm for C3 (cmp. 1A) is not visible in the 13C NMR spectrum.

 Just a hint for future work: I would have recorded an additional HSQC with a smaller spectral width (sw2 40 ppm, o2p 120 ppm, td2 256) just to have a much better resolution in f1. The same I would have done with the HMBC. 

We thank the reviewer for helping us correct the mistake in describing the signals in the NMR spectra.

Although the spectrum is rather complicated, we managed to establish the structure of compound 1.

We agree that the signal at 4.16 ppm for proton H3 (cmp. 1a) looks more like td. We made the necessary corrections in Table 1 and throughout the text of the manuscript.

Althogh the signal at 38.4 ppm for C-3 (cmp. 1A) is not visible in the ¹³C NMR spectrum, we observed it in the HMBC spectrum of compound 1. The reviewer’s comments will be useful for us in our future work.

Have the authors ever tried to record NMR spectra at higher temperature? 

In our opinion, the registration of NMR and ECD spectra for 1 at different temperatures can not significantly clarify the details of 1а ↔ 1b transformation. Such experiments are very informative in the cases, when the compound under study is relatively rigid and there are only one or two large-amplitude motions degrees of freedom (LAM), the movement along which may be treated separately from the movements along other degrees of freedom. Compound 1 belongs to the class of structurally very flexible compounds, for which a big number of LAM motions may proceed at one and the same time (see Figure S71-83, Supplementary data). The IR spectrum calculated theoretically for 1 contains more then 30 vibrational modes with frequencies n £ 350 cm^-1. Fifteen of them have frequencies n £ 150 cm^-1, and for six modes n £ 50 cm^-1. The heating of the sample will lead to the effective growth of populations of the excited states of these modes and, as a result, - to the growth of the so-called “vibrational contribution” to the values of spectral parameters. To extract the contribution of the 1а ↔ 1b transformation from the total experimentally detected temperature dependence one have to use complicated theoretical models, in which the movements along all LAM degrees of freedom are treated simultaneously (with the account of anharmonicity of corresponding potentials). At present such modeling is far and far away from the routine and may be seen as a task for separate investigation.

We recalculate the height of potential barrier for 1а ↔ 1b transformation with 6-311++G(d,p) basis set:

DV^#(B3LYP/6-311++G(d,p)_PCM) » 21 kcal/mol.

This confirms our previous estimation DV^# ³ 20 kcal/mol. The lifetimes of 1a and 1b conformers are t » 3 c for T = 330 K.

This is why we will observe 1a and 1b as two separate compounds in the NMR spectra. Besides, the boiling point of acetone is 56°C (329 K), so we have to record NMR spectra at temperatures lower than 330 K.

It would be a good idea to have the English improved by a native speaker. The main problem besides wrong clause position is the lack of definite and indefinite articles. I mean, article is easy to read and understand, but its not wrong to improve it.

 just some expamples from the first 3 pages

 line 18: stilbens

line 36: is used to produce the drug

line 36: was developed at the G.B. Elyakov Pacific Institute of Bioorganic Chemistry

line 40: dimeric stilbenes [1]. In contrast

line 56: and the isoflavones calicosin and 8-O-methylretusin as  well as the pterocarpan

line 57: The isoflavones genistein 

line 72: properties of the polyphenols from 

With a clear conscience I can propose the manuscript for publication after small revisions.

We made the necessary corrections throughout the text of the manuscript.