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. 2020 Sep 21;15(9):e0238927. doi: 10.1371/journal.pone.0238927

Complexity of active medicinal ingredients in radix scutellariae with sodium hydrosulfite exposure

Ying Shen 1, Wei Cong 1,2, Ai-hua Zhang 1, Xiangcai Meng 1,*
Editor: Branislav T Šiler3
PMCID: PMC7505437  PMID: 32956425

Abstract

Both plants and animals are living things made up of similar cells as well as organelles, and their essence of life is the same. However, plants face more environmental stress than animals and generate excessive reactive oxygen species (ROS), a group of small molecules that can harm proteins, necessitating distinctive metabolic processes. Secondary metabolites in plants are a group of chemical components that can eliminate ROS and can also exhibit medicinal properties; therefore, herbal medicines are often closely linked to the ecological significance of secondary metabolites. Why plants contain so many, not few, active medicinal ingredients is unknown. The root of Scutellaria baicalensis, a popular herbal medicine, is rich in various flavonoids with diverse structural features. Sodium hydrosulfite (Na2S2O4) can produce O˙-2 radicals and induce physical conditions under environmental stress. Using UHPLC-ESI-Q-TOF-MS/MS analysis, a total of 25 different compounds were identified in the roots of S. baicalensis between the Na2S2O4 groups and suitable conditions. Based on the results of the t-test (P<0.05) performed for the groups and ions with values of VIP ≥ 2, the most significantly different chemical markers with Na2S2O4 treatment were shikimic acid, citric acid, baicalin, wogonoside, baicalein, wogonin, 3,5,7,2',6'-pentahydroxyflavanone, 5,2',6'-trihydroxy-7,8-dimethoxy flavone, chrysin, eriodictyol, 5,8-dihydroxy-6,7 -dimethoxy flavone, skullcapflavone Ⅱ, and 5,7-dihydroxy-6,8,2',3'-tetrame thoxyflavone, and most of them were free flavonoids with many phenolic hydroxyl or methoxyl groups and characteristically high antioxidant activities. S. baicalensis roots modified their ability to eliminate ROS and maintained the equilibrium of ROS through the multitudinous biosynthesis and conversion of flavonoids, which is similar to the equilibrium established by an intricate buffer solution and perfectly explains the diversity and complexity of medicinal plant ingredients.

Introduction

Animals can avoid unfavourable circumstances, and the survival of native species is mainly dependent on intraspecific or interspecific competition for foods, not the ecological environment; however, immobile plants can get enough light from the sun, but must face high temperatures, drought, and low soil fertility stress factors, so various adverse effects are almost uninterrupted. Under stress, the light energy absorbed by chloroplasts far outweighs the need to capture CO2, leading to a surfeit of light energy. In addition, closed stomata under stress block O2 emissions outward, and the reduction of O2 to O•-2 (Mehler reaction) is exacerbated [1,2]. O•-2 radicals can be converted further into ·OH and H2O2. These molecules have a strong oxidation power and are described as reactive oxygen species (ROS) that can modify the structures of proteins, including enzymes, by affecting disulfide bridges and regulating various metabolic processes. Therefore, a suitable level of ROS also acts as an indispensable messenger to regulate various physiological actions in plants [3,4], but once ROS are overproduced, a range of destructive forces follow, such as altered adjacent molecular configurations, a reduced cell-membrane stability, DNA strand destruction, protein crosslinking, and peptide chain breakage, resulting in metabolic disorders and even cell death [5]. It has been confirmed that increased ROS levels are a result of stress, resulting in a 3-fold increase in O˙-2 and a 10-fold increase in H2O2 under certain conditions [6]. Under high levels of O˙-2, metabolic alterations under stress can be reproduced; the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) are decreased, and the content of baicalein, an antioxidant compound, was shown to increase from 0.28% to 1.96% [7].

ROS are eliminated mainly by antioxidant enzymes, including SOD, CAT, and POD, and secondary metabolites, including phenolic compounds, carotenoids, and tocopherols. O˙-2 is converted to H2O2 either spontaneously or by SOD and then converted into H2O and O2 by CAT or POD. A suitable level of ROS is an indispensable part of metabolism; however, antioxidant enzymes are also proteins. In particular, some -SH groups that maintain the secondary structure and tertiary structure of the enzymes are liable to be injured if an undue amount of ROS in plants is generated under severe abiotic stresses [5]. Therefore, this might be the reason why plants possess the ability to produce secondary metabolites in addition to antioxidant enzymes.

The secondary metabolites in plants are numerous; by ultra-high-performance liquid chromatography, a total of 132 metabolites in Scutellaria baicalensis, 447 metabolites in Isatis indigotica Fortune, 122 in Moringa oleifera leaves, and 128 in American ginseng roots were identified [8–11], most of which were secondary metabolites. It has been proven that the proportion of secondary metabolites varies according to ever-changing environmental conditions [12]. It is known that the ingredients in herbal medicines are complex, so it is impossible to assess their value by only one or several secondary metabolites. These compounds are usually put into classes with similar structures, whose changes result in variations in their activities [13]. Secondary metabolites are important for plants to adapt to adversity; the reason why plants contain so many, not few, secondary metabolites is unknown. Additionally, the connection between the secondary metabolites is also unknown. S. baicalensis Georgi is distributed throughout semi-arid (steppe) climates and often undergoes severe drought stresses, which are a main factor affecting the biosynthesis of flavonoids [14,15]. To protect themselves under adverse environmental conditions, plants produce various kinds of secondary metabolites, which are also active ingredients exhibiting anti-inflammatory, antitumour, and anti-HIV activities [16].

Metabolomics can acquire comprehensive information about various metabolites by using untargeted biochemical approaches to monitor metabolites and is a new technique used in the field of plant research. Quantitative plant metabolomics, with the ability to improve the comprehensive understanding of plant metabolism under different conditions [17], has been considered the most promising approach for the detection of primary and secondary stress-response metabolites [18]. Since ROS have a dual effect, too much or too little is harmful, and plants require that the equilibrium of ROS is exactly maintained. The large quantities and constantly changing activities of secondary metabolites are perhaps an important tactic for plants to respond to changing environments as quickly and as delicately as possible, which is probably performed through the biosynthesis and conversion of numerous flavonoids for S. baicalensis. O˙-2 is an original ROS that is not present long term in nature due to its poor stability, with a half-life of only approx. 1 μs. Sodium hydrosulfite (Na2S2O4) can produce O˙-2 spontaneously under alkaline conditions [19] and is regarded as an important carrier [20]; Na2S2O4 can easily control O˙-2 production, creating stress physiology. Here, Na2S2O4 was employed to induce stress, and we investigated the different secondary metabolites, the biological significance of flavonoids in S. baicalensis under stress and the diversity and complexity of medicinal ingredients.

Materials and methods

Plant materials and reagents

S. baicalensis Georgi samples were collected from the medical plant garden at Heilongjiang University of Chinese Medicine. In October 2018, 2-year-old plants of S. baicalensis were sprayed with aqueous Na2S2O4 aqueous solutions with concentrations of 40 μmol/L, 0.4 μmol/L, and 0.004 μmol/L, and the control group was kept moist with just water. The samples were collected 0, 1, 2, and 3 days after spraying. A total of 5 plants were selected for each sample, the dirt was washed off, the xylem was removed, and the samples were freeze-dried and then ground into a fine powder.

Methanol (HPLC-grade) was purchased from Fisher Scientific Corporation (Loughborough, UK); HPLC grade acetonitrile was obtained from Merck (Darmstadt, Germany); leucine-enkephalin was purchased from SIGMA Corporation (USA); ultrapure water was produced by a Milli-Q Ultra-pure water purification system (Millipore Corporation, MA, USA). All other reagents were of analytical grade.

Preparation of extracts for UHPLC-ESI-Q-TOF-MS/MS analysis

Collection and preparation of plant samples: Fine powder (150 mg) and 50 mL 70% methanol were placed into conical flasks and ultrasonically extracted for 1 h, and the volume lost was replaced with fresh 70% methanol. Finally, the supernatant was filtered with a 0.22 μm microporous filter for UPLC analysis.

Analytical conditions

Ultra-performance liquid chromatography: Chromatographic separation was performed on an ACQUITY UPLC system (Waters Corporation, Milford, MA) consisting of a binary solvent system, a sample manager and a column compartment. The column used was a UPLCTM BEH C25 column (100 mm× 2.1 mm 1.8 μm, Waters Corporation, Milford, USA). The column temperature was maintained at 40°C for all analyses, and the autosampler temperature was maintained at 10°C. The optimal mobile phase consisted of a linear gradient system of (A) 0.1% formic acid in acetonitrile and (B) 0.1% formic acid in water: 0 to 1.5 min, 16 to 22% A; 1.5 to 5 min, 22 to 30% A; 5 to 9 min, 30 to 40% A; 9 to 12 min, 40 to 70% A; and 12 to 15 min, 70 to 100% A. The detection wavelengths were those used in a full ultraviolet wavelength scan from 190 to 400 nm. The flow rate was set to 0.4 mL/min. The injection volume was 3 μL. The detection of positive and negative ions was performed by the continuous flow of the analytes to the mass spectrometer. All the samples were kept at 4°C during the analysis.

Mass spectrometry

Positive ionization mode: The capillary voltage was 3.0 kV, the sampling cone voltage was 25 V, the extraction voltage was 4.0 V, the desolvation gas temperature was 350°C, the desolvation gas flow was 600 L/h, the source temperature was 110°C, and leucine enkephalin at a concentration of 0.2 ng/mL was used via a lock spray interface and introduced with a flow rate of 100 μL/min for monitoring in positive ionization mode ([M+H]+ = 556.2771) to ensure accuracy during the MS analysis. The lock spray frequency was set to 5 s, and scan averaging for the correction was performed every 0.02 s and 0.4 s. The scanning range was m/z 100~1500.

Negative ionization mode: the capillary voltage was 2.2 kV, the sampling cone voltage was 25 V, the extraction voltage was 3.5 V, the desolvation gas temperature was 350°C, the desolvation gas flow was 600 L/h, the source temperature was 110°C, and leucine enkephalin at a concentration of 0.2 ng/mL was used via a lock spray interface at a flowrate of 100 μL/min for monitoring in negative ionization mode ([M+H]+ = 556.2771) to ensure accuracy during the MS analysis. The lock spray frequency was set to 5 s, and scan averaging for correction was performed every 0.02 s and 0.4 s per scan. The scanning range was m/z 100~1500.

Results

Identification of chemical markers in radix scutellariae

MassLynx V 4.1 was employed for the analysis of the chemical constituents of Radix Scutellariae. The chemical composition was elucidated by the spectral information obtained from secondary ion mass spectrometry, which was cross-referenced with the retention time, mass-to-charge ratio, molecular weight, structural formula and elemental composition of known ingredients in Radix Scutellariae. Based on the VIP results, the different candidate ions observed in the Na2S2O4 treatment groups and the control group were tentatively identified. When taking the identification of baicalin as an example, in positive mode, the ion (RT = 4.92 min and [M+H]+ = 447.12) detected in the Na2S2O4-treated sample was calculated to be C21H19O11 based on the elemental composition, fractional isotope abundance, and Chemspider database information. The main MS/MS fragment ions of peak 9 were m/z 271 and m/z 253, indicating that the fragments may be C15H11O5- and C15H9O4-, which could indicate the loss of glucuronic acid (176 Da) and H2O2 (25 Da), respectively. With this integrated information, the ion was finally confirmed to be that of baicalin (Fig 1). The corresponding mass spectra and related structures are shown in Fig 1. According to the above-mentioned analytical method and relevant literature [8,21], a total of 25 chemical markers that were differentially expressed between the two Radix Scutellariae treatment groups was successfully identified, including 19 candidate ions in positive ion mode and 6 candidate ions in negative mode. Using Waters Masslynx software, we finally confirmed their identities with the MS/MS data. The UPLC-HDMS chromatograms of Scutellaria root (Radix Scutellariae) in positive ion mode and in negative ion mode are shown in Fig 2. Detailed information on all the identified components in different treatments are shown in Table 1.

Fig 1. MS/MS spectrum and the proposed fragmentation pathway of baicalin.

Fig 1

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Fig 2. UPLC chromatograms of Scutellaria root extracts analysed in positive ionization mode and negative ionization mode.

Fig 2

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(a) Positive ion mode; (b) Negative ion mode. A: 0 days; B: 1 day; C: 2 days; D: 3 days; E: 4 days.

Table 1. Characterization of compounds in fresh Scutellaria root extracts by UPLC-HDMS.

Peak No. Rt (min) Selected ion Measured mass (m/z) Calc. mass m/z) Error (ppm) Formula MS/MS fragment ion (m/z) Identification
1 0.54 [M-H]- 173.15 173.03 3.3 C7H11O5 155,137,111 Shikimic Acid
2 0.62 [M-H]- 191.14 191.15 2.9 C6H7O7 173,147 Citric acid
3 2.17 [M+H]+ 305.26 305.21 4.0 C15H13O7 287,153 3,5,7,2',6'-Pentahydroxyflavanone
4 2.8 [M+H]+ 549.15 549.16 1.6 C26H29O13 531,513,495411,375 Aspenin-6-C-Arabinose-8-C-glucoside or Chrysin-6-C-glucose-8-C-Araboside
5 3.19 [M+H]+ 549.15 549.16 0.9 C26H29O13 531,513,495411,375 Aspenin-6-C-Arabinose-8-C-glucoside or Chrysin-6-C-glucose-8-C-Araboside
6 4.16 [M+H]+ 477.10 477.10 2.1 C22H21O12 301,286 5,7,2'-trihydroxy-6-methoxyflavonoid-7-O-glucuronide
7 4.35 [M-H]- 287.26 287.19 2.6 C15H11O6 251,135 Eriodictyol
8 4.56 [M+H]+ 347.07 347.07 4 C17H15O8 332,314 5,7,2',5'- tetrahydroxy -8,6'- dimethyl oxyflavone
9 4.92 [M+H]+ 447.12 447.09 2.5 C21H19O11 271,253 Baicalin
10 5.68 [M+H]+ 447.09 447.09 4 C21H19O11 285 Oroxylin A-5-O glucoside
11 5.83 [M+H]+ 447.09 447.09 4.7 C21H19O11 271 Baicalin isomers
12 5.95 [M+H]+ 477.10 477.10 4.2 C22H21O12 301,286 5,7,8-trihydroxy-6-methoxyflavone-7-O-glucuronide
13 6.19 [M+H]+ 461.10 461.10 4.6 C22H21O11 285,270 Wogonoside
14 6.2 [M-H]- 429.08 429.08 3.5 C21H17O10 253 Chrysin -7-O-glucuronide
15 6.44 [M+H]+ 477.10 477.10 2.9 C22H21O12 301 5,6,7-trihydroxy-8-methoxyflavone-7-O-glucuronide
16 7.01 [M+H]+ 315.30 315.22 1.1 C17H15O6 282,285 5,8-dihydroxy-6,7-dimethoxyflavones
17 7.05 [M-H]- 489.09 489.10 0.8 C23H21O12 313,298 5,7-dihydroxy-6,8-dimethoxyflavone-7-O-glucuronide
18 7.97 [M-H]- 329.88 329.85 3.5 C17H13O7 299 5,2',6'-Trihydroxy-7,8-dimethoxyflavone
19 8.03 [M+H]+ 301.06 301.07 3.7 C16H13O6 286 tenaxin Ⅱ
20 8.21 [M+H]+ 271.05 271.06 3.6 C15H11O5 253,241 Baicalein
21 10.51 [M+H]+ 285.05 285.07 4.6 C16H13O5 270 Wogonin
22 10.67 [M+H]+ 255.06 255.06 4.5 C15H11O4 209 Chrysin
23 11.01 [M+H]+ 375.10 375.18 4.3 C19H19O8 360,345,327 Skullcapflavone II
24 11.05 [M+H]+ 375 375 1.1 C19H19O8 345 5,7-dihydroxy-6,8,2',3'-tetramethoxyflavone
25 11.45 [M+H]+ 345.0 345.09 4.3 C25H17O7 330,315 5,2-dihydroxy-6,7,8-trimethoxyflavones
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With aqueous Na2S2O4 at different concentrations, citric acids presented a downtrend, and the other components exhibited an uptrend, as shown in Fig 3.

Fig 3. Changes in the contents of 13 compounds under Na2S2O4 treatment.

Fig 3

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A: shikimic acid; B: citric acid; C: baicalin; D: wogonoside; E: baicalein; F: wogonin; G: 3,5,7,2',6'-pentahydroxyflavanone; H: 5,2',6'-trihydroxy-7,8-dimethoxyflavone; I: chrysin; J: eriodictyol; K: 5,8-dihydroxy-6,7-dimethoxyflavone; L: Skullcapflavone II; M: 5,7-dihydroxy-6,8,2',3'-tetramethoxyflavone; L: 0.004 μmol/L Na2S2O4; M: 0.4 μmol/L Na2S2O4; H: 40 μmol/L Na2S2O4.

Characteristic multivariate metabolomic data analysis

A PCA model was used to identify the difference in the metabolites of the Na2S2O4 treatment and control groups. The PCA score plots are shown in Fig 4. OPLS-DA was used to discriminate between the groups as well. As shown in Fig 5, the group differences were clearly divided into three regions, indicating that there were significant chemical differences between them, and the established metabolomics method could successfully characterize the chemical characteristics. VIP values are commonly used to evaluate the contribution of variables in OPLS-DA. Based on the results of the t-test (P<0.05) performed for the different groups, ions with VIP values ≥2 were selected and regarded as the most significantly different chemical markers of the Na2S2O4 treatment and control groups. Therefore, shikimic acid, citric acid, baicalin, wogonoside, baicalein, wogonin, 3,5,7,2',6'-pentahydroxyflavanone, 5,2',6'-trihydroxy-7,8-dimethoxy flavone, chrysin, eriodictyol, 5,8-dihydroxy-6,7- dimethoxyflavone, skullcapflavone Ⅱ, and 5,7-dihydroxy-6,8,2',3'-tetramethoxy flavone presented significant differences.

Fig 4. Score plots of UPLC-Q/TOF-MS data generated via PCA in positive ion mode.

Fig 4

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Fig 5. Combination of S-score values of OPLS-DA detected in positive ion mode for the low-Na2S2O4 treatment sample and the control sample on the third day.

Fig 5

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Discussion

Secondary metabolism was enhanced

In this paper, only two primary metabolites were observed in the mass spectral data. Citric acid, a major substance in the tricarboxylic acid cycle, decreased slightly and continuously with increased Na2S2O4 exposure, indicating that its primary metabolism was weakened. Shikimic acid, from which various flavonoids originate, is a branch point of the primary and the secondary metabolic pathways, and the decreased citric acid content and increased shikimic acid content indicated enhanced secondary metabolism, resulting in increased secondary metabolite production; additionally, the content of a total of 13 secondary metabolites increased with the Na2S2O4 treatment. Shikimic acid is located upstream of citric acid, which indicates that more shikimic acid would be converted into secondary metabolites.

Biological significance of varied compounds

Three features of these enhanced secondary metabolites became prominent. First, the molecular structure of the secondary metabolites dictated the biological effect based on the number and sites of the phenolic hydroxyl groups in flavonoids. Flavonoids are a series of compounds containing two benzene rings connected by 3 carbon atoms, and phenolic hydroxyls are a regular moiety on them, as shown in Fig 6. It has been proven that the number of hydroxyl groups on the B ring directly impacts the activity, which is also markedly enhanced when a double bond is introduced into the C ring [22]. The hydroxyl group at positions C-5 and C-7 together in the A ring, as well as the C-3', C-4' and C-5' sites on the B ring, can all increase the activities obviously [23,24]. A hydroxyl group can also be located in another position, such as C-6 [25]. It has been proven that baicalein is 2~5 times more antibiotically active than baicalin and 1~3 times better than baicalin at inhibiting IL-1β and converting enzymes [24]. Second, except for baicalin and wogonoside, the other flavonoids were free flavonoids without sugar moieties. The biosynthesis of flavonoids is performed in the endoplasmic reticulum, and it is difficult for flavonoid glycosides with hydrophilic sugar moieties to freely pass through biomembranes with a lipophilic phospholipid bilayer. The biomembranes of animals and plants are the same; therefore, the biological effect of glycosides would be limited in animals and plants unless the sugar moieties were removed. A study showed that the activities of flavanone disappear when a sugar moiety is introduced into the A ring [22], and another study showed that baicalein is more than 7 times more bioavailable than baicalin due to hydrophilic variations [26]. Flavonoid glycosides therefore are regarded as superfluous flavonoids; when required, they work mainly after their conversion into free flavonoids [27–29]. Third, the contents of some components with methoxy groups increased under high levels of ROS. The methoxy groups on flavonoids have the ability to donate electrons and exhibit a weak antioxidant capacity in their natural state, but their power is remarkably enhanced with the help of peroxidase (POD) [30]. The activity and biosynthesis of POD, an adversity-resistant antioxidase, are induced by ecological stress. Only when the POD activity was high under stress can the components with methoxy groups exert a strong power. These components are usually located at the downstream or terminus of biosynthesised flavonoids and are stable. Therefore, the activities of the components with methoxy groups constantly changed due to ROS formed from ecological stress.

Fig 6. Molecular structure and biosynthesis diagram of flavonoids.

Fig 6

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CHI: chalcone isomerase; FNSII-2: flavone synthase; OMT: O-methyltransferases; FH: flavanone hydroxylase; F6H: flavone 6-hydroxylase; F8H: flavone 8-hydroxylase; UBGAT: glucuronyltransferase; BG:β-glucuronidase.

Interestingly, the above-mentioned secondary metabolites were all highly active at relatively low concentrations, indicating that the effect of compounds with a low concentration cannot be ignored.

Regulation of secondary metabolic pathways

The effects of ROS depend on their concentration in plants, as too much or too little is harmful. Whether there is an overabundance or shortage of ROS depends on the delicate equilibrium between radical production and scavenging [5], which is maintained by antioxidants. Under severe stress, secondary metabolites are highly produced and are coordinated with ROS. The more ROS produced, the higher the flavonoid activity is. The flavonoid activity is regulated through the biosynthesis and interconversion of these flavonoids.

When facing environmental stresses, the increased ROS content swiftly triggered a change in the secondary metabolism. First, the glucuronic acid moieties on baicalin and wogonoside were removed by β-glucuronidase, and baicalin and wogonoside were rapidly converted to baicalein or wogonin as a result of increased activities [31]. Chrysin, with only C-5 and C-7 hydroxyl groups, could also receive a hydroxyl group and was converted into baicalein, norwogonin, and 5,7-dihydroxy-6,8,2',3' -tetramethoxy flavone with more hydroxy groups. The contents of baicalin, wogonoside, and chrysin are all high in the natural state, and furthermore, these reactions are fulfilled by only one step; therefore, the reaction was swiftly performed. Second, ROS can increase the activity of phenylalanine ammonialyase (PAL) [20,32], continuously replenishing flavonoids.

When the ROS content declined, superfluous baicalein or wogonin was converted into baicalin and wogonoside by baicalein7-O-glucuronosyl transferase. The baicalin and wogonoside are confined to certain domains in cells due to hydrophilic glucuronic acid, making it difficult for baicalin and wogonoside to play a role and resulting in a declined antioxidant capacity. In addition, the biosynthesis of flavonoids declined as the level of ROS decreased.

The components with methoxy groups can astutely regulate the antioxidant capacity according to the ROS level, which also plays an important role in maintaining the equilibrium of ROS.

In this paper, only the components with VIP values ≥2 were selected and regarded as the most significant differential chemical markers, and the biosynthesis pathway of 13 different components was summarized, as shown in Fig 6, according to the literature [8,33]. Now, more than 120 components have been reported in S. baicalensis [8], and in reality, the number of activity-relevant pathways must be numerous to maintain the equilibrium of ROS as quickly and delicately as possible.

Conclusion

The diversity of secondary metabolites plays a crucial role in plant bioactivity. A variety of secondary metabolites in S. baicalensis was produced in varying proportions. Under ROS, the contents of free flavonoids with many phenolic hydroxyl groups and characteristically high antioxidant activities increased. The change in the proportion and activities of these compounds modified the ability of the plants to eliminate redundant ROS and maintain ROS equilibrium through the biosynthesis and conversion of flavonoids in a manner similar to that occurring in an intricate buffer solution; in addition, the complexity of the active compounds ensures that plants respond to changing environments as quickly and delicately as possible. Herbal medicines are often closely linked to the ecological significance of secondary metabolites, which perfectly explains the diversity and complexity of medicinal plant ingredients.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors wish to thank for providing financial supports from the National Science Foundation (81573523).

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Decision Letter 0

Branislav T Šiler

29 May 2020

PONE-D-20-06648

C omplexity of A ctive M edicinical I ngredients  in Radix Scutelariae  with Sodium Hydrosulfite Exposure

PLOS ONE

Dear Dr. Shen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript has several shortcomings stated in the reviewers' reports which have to be addressed. In addition to this, my advice to the authors is to thoroughly check the text towards improving overall presentation quality and readability.

Please submit your revised manuscript by Jul 13 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Journal requirements:

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1.    Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitles “Complexity of Active Medicinical Ingredients in Radix Scutelariae with Sodium Hydrosulfite Exposure” was thoroughly reviewed to evaluate its possible acceptance in PLOS One. Overall, the results and discussion of this study are still weak. The manuscript is poorly prepared and required significant improvement. Therefore, unfortunately, this manuscript may not be suitable for publication in PLOS One.

In order to improve the quality of the manuscript, further specific comments are given below, although the list is not exhaustive:

1. First of all, it is very confusing between Scutellaria baicalensis and Radix Scutelariae as the author used in the title and through the text. At least, the authors should be mention Radix Scutelariae is the root of Scutellaria baicalensis, and used one name in all the text.

2. In the abstract and introduction part, the author mention: “The reason why plants contain so many secondary metabolites is unknown” is not exactly. Although we can not discover all the reason, numerous reason for plants contains many secondary metabolites were observed, such as:

- Some of these chemicals enhance their own survival.

- Some plants produce chemicals that act as herbicides to inhibit the growth of competing plants.

- Other plants produce substances that deter browsing by insects and herbivores

- Many plants produced secondary metabolites to respond to environmental stress (biotic, abiotic stress), etc.

3. It is not necessary and too long for explaining bout ROS and antioxidant enzyme in the introduction part. Better to focus on the main content of these results. The role of Na2S2O4 should be deeply mentioned.

4. In material and method: “… 2-year-old plants of S. baicalensis were sprayed with aqueous Na2S2O4 aqueous solutions at concentrations of 40 μmol/L, 0.4 μmol/L, and 0.004 μmol/L, and the control group was kept moist with just water. The samples were collected 0, 1, 2, and 3 days after spraying”. However, the result was not mentioned about the difference of result among those treatments.

5. Which treatment was showen in Table 1?

6. Result and discussion too short to difficult to understand this study.

7. The authors should establish a metabolic synthesis pathway for this plant. It helps explain the result easily.

8. There have no Fig 5L in Fig. 5.

9. All the species name in the references list should be in Italic.

Reviewer #2: manuscript can be accepted after thorough revision keeping in view following points:

1. English writing.

2. cite relevant references.

3. provide some possible mechanisms in the discussion.

4. please rewrite conclusion and make abstract crispy and to the point.

5. a clear hypothesis is missing.

Reviewer #3: Comments

1. In the Abstract of the article, line 8, it is better for the authors to add the Scutellaria inside the parentheses after the root of Scutellaria, and use it later in the whole article.

2. The authors used the Na2SO4 compound to induce oxidative treatment. What is the reason for using this compound? Explain in the article

3. “Sodium Dithionite-Enhanced Quality of Scutellaria Through Modification of Secondary Metabolism” in the introduction, the last paragraph, the reference should be given.

4. In the materials section, ethanol solvents are used for extraction, while methanol solvents are commonly used to extract flavonoids. What is the reason for use? Explain the reason in manuscript

Results

5. In Table 1, the acronym” m / z “ should be written in Italic

6. In the table in the column related to MS / MS fragment ion, for the compounds that have been identified in both positive and negative ionization methods, the negative ionization pattern is also should be written.

7. In Table 4, combinations 4 and 5 both have the same negative and positive ionization modes, the same m / z, and the same ionization pattern have considered while they did not use any standard combinations.

8. In Table 1, the authors include 25 compounds. Are these compounds first identified from this plant? If not, it is best to briefly explain the composition of each compound and add the relevant references.

9. In Figure 2, the chromatogram images please show the root extract in the positive and negative ionization state of the peak corresponding to each combination with numbers.

Discussion

10. According to statistical analysis, compounds whose VIP is larger or equal to 2 (VIP ≥2) have been selected for further study and the number of these compounds is 13, while in the discussion section of the first paragraph, 12 compounds are mistakenly written. That needs to be edited.

11. In the discussion section of the last paragraph, it is better for the authors to give a brief explanation about the path of biosynthesis of flavonoids in the roots of Scutellaria.

12. In the last paragraph of the last line, in explaining Figure 5, we talk about the multiplicity of biosynthetic pathways of flavonoid compounds, while Figure 5 does not. Figure 5 provides information on the content of compounds identified under different doses of oxidative treatment, and no such connection is made.

13. In Figure 5, it is best to add a chart of the contents of the studied compounds to the image in control mode. The values provided are for treatment only

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Ali Sharafi

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Reviewer comment_PONE-D-20-06648.docx

Click here for additional data file. (19.8KB, docx)
PLoS One. 2020 Sep 21;15(9):e0238927. doi: 10.1371/journal.pone.0238927.r002

Author response to Decision Letter 0

3 Jul 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitles “Complexity of Active Medicinical Ingredients in Radix Scutelariae with Sodium Hydrosulfite Exposure” was thoroughly reviewed to evaluate its possible acceptance in PLOS One. Overall, the results and discussion of this study are still weak. The manuscript is poorly prepared and required significant improvement. Therefore, unfortunately, this manuscript may not be suitable for publication in PLOS One.

Answer: Thank you for your suggestion. This manuscript aim to clarify why medicine plants contain so many active medicinical ingredients in the perspective of ecological stress. The discussion part were supplemented and throughly revised.

In order to improve the quality of the manuscript, further specific comments are given below, although the list is not exhaustive:

1. First of all, it is very confusing between Scutellaria baicalensis and Radix Scutelariae as the author used in the title and through the text. At least, the authors should be mention Radix Scutelariae is the root of Scutellaria baicalensis, and used one name in all the text.

Answer: Radix Scutelariae is the root of Scutellaria baicalensis, to avoid confusing, the difference between them was labeled at starting part.

2. In the abstract and introduction part, the author mention: “The reason why plants contain so many secondary metabolites is unknown” is not exactly. Although we can not discover all the reason, numerous reason for plants contains many secondary metabolites were observed, such as:

- Some of these chemicals enhance their own survival.

- Some plants produce chemicals that act as herbicides to inhibit the growth of competing plants.

- Other plants produce substances that deter browsing by insects and herbivores

- Many plants produced secondary metabolites to respond to environmental stress (biotic, abiotic stress), etc.

Answer: It is a problem coming from confussing expression! We mean to express the reason that plants contain so many secondary metabolites, can a few kinds of secondary metabolites with similiar effects do that? Based on this hypothesis, complexity of Active Medicinical Ingredients in Radix Scutelariae can be pesented. The manuscript was revised in another words.

3. It is not necessary and too long for explaining bout ROS and antioxidant enzyme in the introduction part. Better to focus on the main content of these results. The role of Na2S2O4 should be deeply mentioned.

Answer: This article aim to expound the complexity of active medicinical ingredients in Radix Scutelariae, nevertheless, active medicinical ingredients are often closely linked to the ecological stress, to which ROS and antioxidant enzymes can adjust automatically. It is probably caused by poorly expression, have been revised in article.

4. In material and method: “… 2-year-old plants of S. baicalensis were sprayed with aqueous Na2S2O4 aqueous solutions at concentrations of 40 μmol/L, 0.4 μmol/L, and 0.004 μmol/L, and the control group was kept moist with just water. The samples were collected 0, 1, 2, and 3 days after spraying”. However, the result was not mentioned about the difference of result among those treatments.

Answer: With aqueous Na2S2O4 at different concentrations, Citric acid presented a downtrend, the other ingredient a uptrend, as shown in Fig. 3.

5. Which treatment was showen in Table 1?

Answer: The table. 1 was all the identified components in different treatments, which has been further clarificated.

6. Result and discussion too short to difficult to understand this study.

Answer: A pertinent suggestion! With aqueous Na2S2O4 at different concentrations, The change trends has been added in Result part. The pathway of biosynthesis and transformation of secondary metabolites has been supplemented.

7. The authors should establish a metabolic synthesis pathway for this plant. It helps explain the result easily.

Answer: Good idea! The pathway of biosynthesis and transformation of secondary metabolites has been supplemented.

8. There have no Fig 5L in Fig. 5.

Answer: it is mistaken written. It has been revised.

9. All the species name in the references list should be in Italic.

Answer: It has been revised.

Reviewer #2: manuscript can be accepted after thorough revision keeping in view following points:

1. English writing.

Answer: The manuscript has polished by English Editing Services nominated by PLOS ONE, as shown in attachment. If need to further improved, we will contact them.

2. cite relevant references.

Answer: It has been seriously revised according to PLOS ONE.

3. provide some possible mechanisms in the discussion.

Answer: The discussion part was supplemented many information and throughly revised. The pathway of biosynthesis and transformation of secondary metabolites has been supplemented, and further provided some possible mechanisms.

4. please rewrite conclusion and make abstract crispy and to the point.

Answer: It is a good suggestion and has been revised.

5. a clear hypothesis is missing.

Answer: It has been supplemented at last part in Introduction.

Reviewer #3: Comments

1. In the Abstract of the article, line 8, it is better for the authors to add the Scutellaria inside the parentheses after the root of Scutellaria, and use it later in the whole article.

Answer: Good idea, it has been supplemented.

2. The authors used the Na2SO4 compound to induce oxidative treatment. What is the reason for using this compound? Explain in the article

Answer: It has been supplemented at last part in Introduction.

3. “Sodium Dithionite-Enhanced Quality of Scutellaria Through Modification of Secondary Metabolism” in the introduction, the last paragraph, the reference should be given.

Answer: It is a good suggestion. It has been supplemented.

4. In the materials section, ethanol solvents are used for extraction, while methanol solvents are commonly used to extract flavonoids. What is the reason for use? Explain the reason in manuscript

Answer: The methanol solvents is right, the ethyl alcohol was mistakenly written.

Results

5. In Table 1, the acronym” m / z “ should be written in Italic

Answer: All acronym “m / z” in muanuscript has been revised into “m / z” .

6. In the table in the column related to MS/MS fragment ion, for the compounds that have been identified in both positive and negative ionization methods, the negative ionization pattern is also should be written.

Answer: Thank you for your suggestion, we have revised it in the manuscript.

7. In Table 4, combinations 4 and 5 both have the same negative and positive ionization modes, the same m / z, and the same ionization pattern have considered while they did not use any standard combinations.

Answer: The combinations 4 and 5 both have the same negative and positive ionization modes, the same m / z, and the same ionization pattern, they must be isomers. It is reqired standard combinations to identify them, but we have no way to obtain them, so, in article it was revised into “Aspenin-6-C-Arabinose-8-C-glucoside or Chrysin- 6-C- glucose - 8 - C - Araboside”.

8. In Table 1, the authors include 25 compounds. Are these compounds first identified from this plant? If not, it is best to briefly explain the composition of each compound and add the relevant references.

Answer: These compounds first identified are from Scutellaria baicalensis, the relevant references has been add.

9. In Figure 2, the chromatogram images please show the root extract in the positive and negative ionization state of the peak corresponding to each combination with numbers.

Answer: Thank you for your suggestion, we have revised it in the manuscript.

Discussion

10.According to statistical analysis, compounds whose VIP is larger or equal to 2 (VIP ≥2) have been selected for further study and the number of these compounds is 13, while in the discussion section of the first paragraph, 12 compounds are mistakenly written. That needs to be edited.

Answer: It was mistaken, has been revised.

11. In the discussion section of the last paragraph, it is better for the authors to give a brief explanation about the path of biosynthesis of flavonoids in the roots of Scutellaria.

Answer: It is a good suggestion, has been supplemented.

12. In the last paragraph of the last line, in explaining Figure 5, we talk about the multiplicity of biosynthetic pathways of flavonoid compounds, while Figure 5 does not. Figure 5 provides information on the content of compounds identified under different doses of oxidative treatment, and no such connection is made.

Answer: It has been revised.

13. In Figure 5, it is best to add a chart of the contents of the studied compounds to the image in control mode. The values provided are for treatment only

Answer: The values have been provided in Fig.3.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file. (39.5KB, doc)

Decision Letter 1

Branislav T Šiler

20 Jul 2020

PONE-D-20-06648R1

Complexity of Active Medicinal Ingredients in Radix Scutelariae  with Sodium Hydrosulfite Exposure

PLOS ONE

Dear Dr. Shen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please see my comments below, under Additional Editor Comments.

==============================

Please submit your revised manuscript by Sep 03 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Abstract lacks flow. There is no logical connection among sentences. In needs to be rewritten.

The whole text contains vernacular expessions and phrases which cannot be accepted to be published in a scientific journal. Therefore, I strongly encourage the authors to ask a senior scientist to read the manuscript and unbiasedly suggest corrections.

Expressions are randomly capitalized.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript was thoroughly reviewed to evaluate its possible acceptance in Plos One. The authors carefully revised and improved all the addressed points commented by the reviewers. The manuscript can be accepted for publication.

Reviewer #3: The authors made their manuscript acceptable for publication and No additional comments are needed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Thanh-Tam Ho

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Sep 21;15(9):e0238927. doi: 10.1371/journal.pone.0238927.r004

Author response to Decision Letter 1

11 Aug 2020

Thank you for your suggestion. The fluidity and logical relationship of the abstract in the manuscript have been revised, and AJE experts have been invited to revise the nonstandard sentences in the whole manuscript, Editing Certificate is included in the attachment. Thank you.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file. (11.5KB, doc)

Decision Letter 2

Branislav T Šiler

13 Aug 2020

PONE-D-20-06648R2

Complexity of Active Medicinal Ingredients in Radix Scutelariae  with Sodium Hydrosulfite Exposure

PLOS ONE

Dear Dr. Shen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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The authors are advised to meticulously check the text again in order to rectify vague and non-scientific expressions such as "exposed to either Na2S2O4 or suitable conditions" (Abstract). 

Moreover, the text still contains sentences which lack scientific sense such as "...the most significantly different chemical markers of Na2S2O4 were shikimic acid, citric acid..." (Abstract) - I can hardly believe that sodium hidrosulfite can have chemical markers.

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PLoS One. 2020 Sep 21;15(9):e0238927. doi: 10.1371/journal.pone.0238927.r006

Author response to Decision Letter 2

24 Aug 2020

Thank you for your suggestion. There are indeed vague and non-scientific expressions in the abstract section of the manuscript. We have revised them. Thank you again for your suggestions.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file. (11.5KB, doc)

Decision Letter 3

Branislav T Šiler

27 Aug 2020

Complexity of Active Medicinal Ingredients in Radix Scutelariae  with Sodium Hydrosulfite Exposure

PONE-D-20-06648R3

Dear Dr. Shen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Branislav T Šiler

2 Sep 2020

PONE-D-20-06648R3

Complexity of Active Medicinal Ingredients in Radix Scutellariae with Sodium Hydrosulfite Exposure

Dear Dr. Shen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Branislav T. Šiler

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Reviewer comment_PONE-D-20-06648.docx

    Click here for additional data file. (19.8KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.doc

    Click here for additional data file. (39.5KB, doc)
    Attachment

    Submitted filename: Response to Reviewers.doc

    Click here for additional data file. (11.5KB, doc)
    Attachment

    Submitted filename: Response to Reviewers.doc

    Click here for additional data file. (11.5KB, doc)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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