Review of Constituents and Biological Activities of Triterpene Saponins from Glycyrrhizae Radix et Rhizoma and Its Solubilization Characteristics

Round 1

Reviewer 1 Report

The manuscript provides a review in the constituents of triterpene saponins from Glycyrrhizae Radix et Rhizoma and related bioactive properties. It provides considerable information and it is well written and organized with numerous references to previous studies. For further publication, I would just suggest minor modifications, as follows:

The sentence starting at line 169 (“At present, the world is facing the Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). GA (1) could be considered as one of the best constituents which could prove useful against COVID-19.”) should be moved to the conclusions as an example of future works to be considered with these plants, but it remains a speculation since there are no evidences that support its efficacy.

The fact that Figure 1 and 2 have the same title is quite confusing, the authors should consider writing it differently.

In line 209, please substitute “further explaining the” with “further explaining that the”.

 

Author Response

1. The sentence starting at line 169 (“At present, the world is facing the Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). GA (1) could be considered as one of the best constituents which could prove useful against COVID-19.”) should be moved to the conclusions as an example of future works to be considered with these plants, but it remains a speculation since there are no evidences that support its efficacy.

Thank you for your thoughtful suggestions. Surprisingly, in the course of the manuscript revision, we found that there was a new report which could prove that Glycyrrhizin (GL) is useful against COVID-19. So we have revised the sentence “At present, the world is facing the Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). GA (1) could be considered as one of the best constituents which could prove useful against COVID-19.” to “At present, the world is facing the Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). GL (1) has been used to control COVID-19 infections, which may reduce the severity of an infection with COVID-19 at the two stages of the COVID-19 induced disease process, 1. To block the number of entry points and 2. provide an angiotensin converting enzyme 2 (ACE2) independent anti-inflammatory mechanism.” at line 170 in revised manuscript.

The followed content is for your reference only:

The reference is:

[57] Murck, H. Symptomatic protective action of Glycyrrhizin (Licorice) in COVID-19 Infection? Front Immunol. 2020, 11, 1239. DOI: 10.3389/fimmu.2020.01239.

And the detailed content in the reference is:

Glycyrrhizin (GL) is a frequent component in traditional Chinese medicines, which have been used to control COVID-19 infections. Its systemically active metabolite glycyrrhetinic acid (GA) inhibits 11beta hydroxysteroid dehydrogenase(11betaHSD2) and activates MR in organs, which express this enzyme, including the lungs. Does this affect the protective effect of ACE2? Importantly, GL has anti-inflammatory properties by itself via toll like receptor 4 (TLR4) antagonism and therefore compensates for the reduced protection of the downregulated ACE2. Finally, a direct effect of GL or GA to reduce virus transmission exists, which may involve reduced expression of type 2 transmembrane serine protease (TMPRSS2), which is required for virus uptake. Glycyrrhizin may reduce the severity of an infection with COVID-19 at the two stages of the COVID-19 induced disease process, 1. To block the number of entry points and 2. provide an ACE2 independent anti-inflammatory mechanism.

2. The fact that Figure 1 and 2 have the same title is quite confusing, the authors should consider writing it differently.

Thanks for your detailed suggestions. We have revised “Figure 1. Triterpene saponins in G. uralensis.” to “Figure 1. Triterpene saponins in G. uralensis. (A)” in line 101, and “Figure 2. Triterpene saponins in G. uralensis.” to “Figure 2. Triterpene saponins in G. uralensis. (B)” in line 104.

3. In line 209, please substitute “further explaining the” with “further explaining that the”.

We have revised the “further explaining the” to “further explaining that the” in line 209.

Reviewer 2 Report

The work arises from the clever idea such of the possibility of using these saponins as natural surfactants. From this statement just in the introduction the paper  invites to be read with curiosity. The conclusions are interesting. From my point of view the work has enough potential to be published in a magazine like  Molecules,but it must be subject to a major order review previously.

I suggest the following modifications:

• Line 56. Add a paragraph describing how the literature review that has been  submittedhas beenperformed : consulted databases, evaluated period,  keywords used for the search. Table 1 could be improved by making the following changes:
  • Eliminate The Columns of Chemical Formula and Molecular Weight, because they do not provide meaningful information
  • Replace the Origin scientific names of species in the Origin Column with numerical codes  1, 2, 3,4,4
  • Include a column called Structure and put in each case the acronym with which the structure appears in Figures 1, 2, 3,
  • Reread the text after having Table 1 modified, and review whether any comments on it  need to be modified, or if any columns can be added to Table 1 so that it is even more informative and better. TRY TO MAKE EVERYTHING SAID IN THE TEXT GRAPHICALLY EXPRESSED IN TABLE 1, IN AN EASY AND QUICK WAY TO UNDERSTAND FOR THE READER.

1.  

• Line 117.
  • Add a Table 2, which includes the following columns: 1.No, 2.Triterpenoid, 3.Activity 4. Reference
  • Columns 1 and 2 shall be the same in Table 1. If many rows in Table 2 are empty, they can be grouped together.
  • Column 3 can be subdivided into more columns if the final result is found to be more didactic by breaking down that column.
  • Reread the text after having Table 2, following the same guidelines as specified for Table 1.
  • Lines 201-204, should be  included in the Introduction
• Line 208.Table 3 should be included where the contents of paragraphs 3.1 and 3.2 are summarized.

Author Response

1. Line 56. Add a paragraph describing how the literature review that has been submitted has been performed: consulted databases, evaluated period, keywords used for the search. Table 1 could be improved by making the following changes:

Eliminate The Columns of Chemical Formula and Molecular Weight, because they do not provide meaningful information

Replace the Origin scientific names of species in the Origin Column with numerical codes 1, 2, 3, 4

Include a column called Structure and put in each case the acronym with which the structure appears in Figures 1, 2, 3, 4

Reread the text after having Table 1 modified, and review whether any comments on it need to be modified, or if any columns can be added to Table 1 so that it is even more informative and better. TRY TO MAKE EVERYTHING SAID IN THE TEXT GRAPHICALLY EXPRESSED IN TABLE 1, IN AN EASY AND QUICK WAY TO UNDERSTAND FOR THE READER.

Thanks for your significant suggestions, we have added a paragraph describing “SciFinder, PubMed, Web of Science, China Journal Net, and relevant English and Chinese literature were used as information sources by the inclusion of the primary search terms ‘Glycyrrhizae Radix’, ‘glycyrrhiza’, ‘Gan-Cao’, ‘liquorice’, ‘licorice’, ‘triterpene saponins’, ‘constituent’, ‘glycyrrhizin’, ‘glycyrrhizic acid’, ‘activities’, ‘solubilization’, and their combinations, mainly from 1984 to 2020.” in line 57 in the revised manuscript according to your modification suggestion.

Ö Table 1 has been revised including eliminating The Columns of Chemical Formula and Molecular Weight, replacing the Origin scientific names of species in the Origin Column with the little English letters a, b, c, instead of the numerical codes 1, 2, 3, 4, because they had been used in the serial number of Table 1.

After days of reflection, we didn’t insert a column called Structure because a lot of the acronym with the structure are the same. We are so sorry that we haven’t thought of a good way to express structure in Figures 1, 2, 3, 4 so far. Then Table 1 has been revised as follows:

No.	Compound	Origin	References
1	glycyrrhizin (glycyrrhizic acid, uralsaponin A or 18β-glycyrrhizic acid)	a, b, c	16 17 18 19 20
2	uralsaponin B	a, b	16 21
3	licorice-saponin A3	a, c	20 22
4	licorice-saponin B2	a, b	22 23
5	licorice-saponin C2	a, b	22 23
6	licorice-saponin D3	a	22
7	licorice-saponin E2	a, c	20 22
8	licorice-saponin F3	a	24
9	licorice-saponin G2	a, b, c	20 23 24
10	licorice-saponin H2	a, b	23 24
11	licorice-saponin J2	a, b	23 24
12	licorice-saponin K2	a, b	21 24
13	licorice-saponin L3	a	25
14	18α-glycyrrhizic acid	a, b	17
15	apioglycyrrhizin	b, c	23 26
16	araboglycyrrhizin	a, b, c	23 26 27
17	22β-acetoxylglycyrrhizin	a, c	20 28
18	3β-O-[β-D-glucuronopyranosyl- (1→2)-β-D-glucuronopyranosyl]-glycyrretol	a	28
19	3β-O-[β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl]- olean-9,12-diene-30-oic acid	a	28
20	uralsaponin C	a	29
21	uralsaponin D	a, c	20 29
22	uralsaponin E	a	29
23	uralsaponin F	a	29
24	3-O-[β-D-glucuronopyranosyl- (1→2)-β-D-galactopyranosyl]glycyrrhetic acid	a	30
25	licorice-saponin M3 (uralsaponin T)	a, b	27 31
26	licorice-saponin N4	a, b	31 32
27	licorice-saponin O4	b	31
28	uralsaponin M	a	27
29	uralsaponin N	a	27
30	uralsaponin O	a	27
31	uralsaponin P	a	27
32	uralsaponin Q	a	27
33	uralsaponin R	a	27
34	uralsaponin S	a	27
35	uralsaponin U	a	27
36	uralsaponin V	a, b	21 27
37	uralsaponin W	a	27
38	uralsaponin X	a	27
39	uralsaponin Y	a	27
40	22β-acetoxyl-glycyrrhaldehyde	a, c	20 33
41	3-O-β-D-glucuronopyranosyl-glycyrrhetinic acid	a, b	21 27
42	3-O-[β-D-(6-methyl)glucuro-nopyranosyl (1→2)-D-glucurono-pyranosyl]-24-hydroxyglabrolide	a	34
43	licorice-saponin P2	c	20
44	licorice-saponin Q2	c	20
45	macedonoside A	b, c	20 21
46	24-hydroxy-licorice-saponin E2	c	20
47	macedonoside E	a	35
48	22β-acetyl-uralsaponin C	a	35
49	licorice saponin M1	b	21
50	licorice saponin M2	b	21
51	licorice saponin M3	b	21
52	licorice saponin M4	b	21
53	30-hydroxyglycyrrhizin	b	23
54	glycyrrhizin-20-methanoate	b	23
55	24-hydroxyglucoglycyrrhizin	b	23
56	rhaoglycyrrhizin	b	23
57	11-deoxorhaoglycyrrhizin	b	23
58	rhaoglucoglycyrrhizin	b	23
59	rhaogalactoglycyrrhizin	b	23
60	11-deoxo-20α- glycyrrhizin	b	23
61	20α-galacturonoylglycyrrhizin	b	23
62	20α-rhaoglycyrrhizin	b	23
63	glyuralsaponin A	a	32
64	glyuralsaponin B	a	32
65	glyuralsaponin C	a	32
66	glyuralsaponin D	a	32
67	glyuralsaponin E	a	32
68	glyuralsaponin F	a	32
69	glyuralsaponin G	a	32
70	glyuralsaponin H	a	32
71	glabasaponin A	b	36
72	glabasaponin B	b	36
73	glabasaponin C	b	36
74	glabasaponin D	b	36
75	glabasaponin E	b	36
76	glabasaponin F	b	36
77	glabasaponin G	b	36

a, G. uralensis Fisch.; b, G. glabra L.; c, G. inflate Bat.

We have carefully read the text to modify it as following:

(1) The sentence “Notably, some glabrolides (7, 21, 22, 30, 39, 42, and 46) were found in G. uralensis and G. inflate,” has been revised to “Notably, some glabrolides (7, 21, 22, 30, 39, 42, and 46) were found in G. uralensis or/and G. inflate,”

(2) The structure of compound 27 in Figure 1 has been deleted.

(3) The structure of compound 8 in Figure 2 has been added.

(4) The structure of compound 1 in Figure 3 has been added.

 

2. Line 117.

Add a Table 2, which includes the following columns: 1. No, 2. Triterpenoid, 3. Activity 4. Reference

Columns 1 and 2 shall be the same in Table 1. If many rows in Table 2 are empty, they can be grouped together.

Column 3 can be subdivided into more columns if the final result is found to be more didactic by breaking down that column.

Reread the text after having Table 2, following the same guidelines as specified for Table 1.

Thank you for your thoughtful detailed suggestions. According to your modification suggestion, we had added Table 2 in line 142 in revised manuscript and modified seriously the text. The modified text has been used the "Track Changes" function in Microsoft Word. Table 2 has been added as follows:

 

Table 2. Summary of the biological activities conducted with triterpene saponins in glycyrrhiza ¹

No.	Compound	Activity			References
		Property	Method	Major findings
1	glycyrrhizin (glycyrrhizic acid, uralsaponin A or 18β-glycyrrhizic acid)	Hepatoprotective activities	In vitro - primary rat hepatocytes injured by D-galactosamine (D-GalN)	Lower ALT and AST levels	20
			PLA2 inhibitory potency	IC50 = 9.3 μM	20
			In vitro - primary cultured rat hepatocytes induced by CCl4	Prevent soluble enzyme release	39
			In vitro - PLC/PRF/5 cells	Modify the expression of HBV-related antigens on the hepatocytes and suppress sialylation of HBsAg	40
			In vivo - BALB/c mice	Suppress increases in AST and ALT, inhibite iNOS mRNA expression, and reduce protein and cell infiltration and the degeneration of hepatocytes	41
			In vivo - ICR mice	Alleviate CCl4-induced liver injury	42
			In vivo - Sprague Dawley rats	Exhibit protective effect on retrorsine-induced liver damage	43
			In vivo - Wistar rats	Provide partial protection of the liver against ischemia-reperfusion damage	44
			In vivo - Wistar rats	Protect against NTiO2-induced hepatotoxicity	45
		Anti-inflammatory activities	In vitro - LPS-stimulated MEEC	Inhibit LPS-induced inflammatory response by inhibiting TLR4 signaling pathway	47
			In vitro - neutrophil	Inhibit ROS generation by neutrophils	48
			In vivo - Sprague Dawley rats	Inhibit HMGB1 expression and subsequent production of inflammatory cytokines to prevent CVS following SAH	49
			In vivo – SD rats	Alleviate brain injury after DAI via its anti-inflammatory effects	50
		Antimicrobial and antiviral activities	In vitro	Inhibit VZV	53
			In vitro	Inhibit SARS-CoV replication	54
			In vitro	Inhibited IAV uptake into the cell	55
			In vitro	Reduce the severity of an infection with COVID-19 at the two stages of the COVID-19 induced disease process, 1. To block the number of entry points and 2. provide an ACE2 independent anti-inflammatory mechanism.	57
			The commercial NA inhibitory screening kit	Possess moderate influenza NA inhibitory activity	31
		Cytotoxic and antitumor activities	In vivo –tumor-bearing mice	Reduce expression of TxAS, as well as PCNA, and rescue liver and kidney damage	58
			In vitro- HepG2	Display protective effects against AFB1 induced cytotoxicity	59
		Other activities	-	1. Possess immunomodulatory, neuroprotective effects, and antioxidant activities; 2. Bronchitis, peptic ulcers, skin diseases, and oral diseases; 3. Allergic rhinitis	56, 61-66
3	licorice-saponin A3	Antimicrobial and antiviral activities	The commercial NA inhibitory screening kit	Possess moderate influenza NA inhibitory activity	31
9	licorice-saponin G2	Hepatoprotective activities	In vitro - primary rat hepatocytes injured by D-galactosamine (D-GalN)	Lower ALT and AST levels	20
			PLA2 inhibitory potency	IC50 = 16.9 μM	20
		Antimicrobial and antiviral activities	The commercial NA inhibitory screening kit	Possess moderate influenza NA inhibitory activity	31
17	22β-acetoxylglycyrrhizin	Hepatoprotective activities	In vitro - primary rat hepatocytes injured by D-galactosamine (D-GalN)	Lower ALT and AST levels	20
			PLA2 inhibitory potency	IC50 = 27.1 μM	20
		Antimicrobial and antiviral activities	In vitro - MDCK cells	Inhibit influenza virus A/WSN/33 (H1N1)	27
			The commercial NA inhibitory screening kit	Possess moderate influenza NA inhibitory activity	31
21	uralsaponin D	Hepatoprotective activities	PLA2 inhibitory potency	IC50 = 32.2 μM	20
25	licorice-saponin M3 (uralsaponin T)	Antimicrobial and antiviral activities	In vitro - MDCK cells	Inhibit influenza virus A/WSN/33 (H1N1)	27
			The commercial NA inhibitory screening kit	Possess moderate influenza NA inhibitory activity	31
28-39	uralsaponins M-Y	Antimicrobial and antiviral activities	In vitro - MDCK cells	Uralsaponin M (28) and uralsaponin S (34) exhibited inhibitory activities against influenza virus A/WSN/33 (H1N1)	27
44-45	licorice-saponin Q2 (44) macedonoside A (45)	Hepatoprotective activities	In vitro - primary rat hepatocytes injured by D-galactosamine (D-GalN)	Lower ALT and AST levels	20
			PLA2 inhibitory potency	IC50 = 3.6 μM (44) and 6.9 μM (45)	20
63-70	glyuralsaponins A-H	Hepatoprotective activities	MDA colorimetric assay	Glyuralsaponin B (64) and glyuralsaponin H (70) exhibited moderate antioxidant activities against Fe2+/cysteine-induced liver microsomal lipid peroxidation	32

¹, Columns 1 and 2 is the same in table 1. It should be noted that the empty rows with triterpene saponins are grouped together or not listed in Table 2.

 

 

 

3. Lines 201-204, should be included in the Introduction.

We have modified the text in lines 201-204 to “In nature, saponins are distributed in 90 plant families from 500 genera [67]. Some of them have the potential to be used as natural surfactants because they contain the nonpolar sapogenin and water-soluble sidechain [68]. Glycyrrhiza is the most frequently used TCM in TCM formulae, with the function of harmonizing all kinds of TCMs. Researches on the chemistry, pharmacological effects, clinical applications et al of glycyrrhiza has been very extensive in the last few decades. Besides, the saponins from glycyrrhiza have also significant solubilizing effects [69]. Interestingly, the solubilization characteristics of glycyrrhiza and saponins from glycyrrhiza were studied extensively over recent years. This part of review will deal with the solubilization characteristics, influences, and mechanisms regarding glycyrrhiza and triterpene saponins from glycyrrhiza.” in lines 218-226 in the revised manuscript.

 

4. Line 208. Table 3 should be included where the contents of paragraphs 3.1 and 3.2 are summarized.

Thanks for your significant suggestions. As advised, we had added Table 3 in line 224 in revised manuscript. Table 3 has been added as follows:

Table 3. Summary on solubilization of glycyrrhiza and GL (1)

No.	Name	TCM formulae/ TCM/Component	Characteristics	Major findings	References
1	glycyrrhiza	sijunzi decoction, huangqi dazao decoction, baishao gancao decoction	Glycyrrhiza has solubilization effects in three TCM formulae	The solubilizing components in glycyrrhiza are triterpene saponins	70
2		ephedra	The contents of GL (1), ephedrine, and methephedrine et al all increase	-	71
3		epimedium	Icariin in epimedium increases	-	72
4		curcuma longa	The extractive rate of curcumin double	GL (1) is the main surfactant	73
5		codonopsis, poria, atractylodes, Baphicacanthus cusia, Paeoniae Radix Alba, Isatidis Radix, and Scutellaria baicalensis	Glycyrrhiza can increase the contents of active ingredients in these TCM	GL (1) is the main surfactant	74-78
6		Schisandra chinensis	No solubilization effects	One of the possible factors affecting the solubilization is some other crude drugs	70
7	GL (1)	saikosaponin-a	The contents of saikosaponin-a increase	Solubilizing effect is due to GL (1)	83
8		Ben Lamge granules	The solubility of Ben Lamge granules increases	1. The surface tension of GL (1) decreases; 2. GL (1) exists in micelles in aqueous solution.	82
9		baicalin	The dissolution rate of baicalin increases	-	84
10		pueraria	The solubility of pueraria increases	Another possible factors affecting the solubilization is the pH value of the solution	85
11		pachymic acid	Increase the solubility of pachymic acid	Improve the bioavailability of pachymic acid	69
12		puerarin–glycyrrhizic acid dispersible tablets	Improve the dissolution of puerarin	GL (1) possesses solubilization effect	86

 

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

_{Corrections hace been included, so I agree with the acceptance of the paper.}