Investigating the active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald injuries

Yanlin Zou; Cao Yu; Qian Huang; Xiaorong Tan; Xiaoyan Tan; Xiaolong Zhu; Dongyang Yi; Jingxin Mao

doi:10.1371/journal.pone.0287147

. 2023 Jun 13;18(6):e0287147. doi: 10.1371/journal.pone.0287147

Investigating the active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald injuries

Yanlin Zou ^1,^2,³, Cao Yu ^1,³, Qian Huang ^1,³, Xiaorong Tan ^1,³, Xiaoyan Tan ^1,³, Xiaolong Zhu ^1,³, Dongyang Yi ^1,^3,^*, Jingxin Mao ^2,^4,^*

Editor: Sairah Hafeez Kamran⁵

PMCID: PMC10263327 PMID: 37310979

Abstract

Objective

To identify the most effective fraction of Nanocnide lobata in the treatment of burn and scald injuries and determine its bioactive constituents.

Methods

Chemical identification methods were used to analyze solutions extracted from Nanocnide lobata using petroleum ether, ethyl acetate, n-butanol using a variety of color reactions. The chemical constituents of the extracts were identified by ultra-performance liquid chromatography (UPLC)–mass spectrometry (MS). A total of 60 female mice were randomly divided into the following 6 groups: the petroleum ether extract-treated group; the ethyl acetate extract-treated group; the n-butanol extract-treated group; the model group; the control group; and the positive drug group. The burn/scald model was established using Stevenson’s method. At 24 hours after modeling, 0.1 g of the corresponding ointment was evenly applied to the wound in each group. Mice in the model group did not undergo treatment, while those in the control group received 0.1 g of Vaseline. Wound characteristics, including color, secretions, hardness, and swelling, were observed and recorded. Photos were taken and the wound area calculated on the 1^st, 5^th, 8^th, 12^th, 15^th, 18^th and 21^st days. Hematoxylin-eosin (HE) staining was utilized to observe the wound tissue of mice on the 7^th, 14^th, and 21^st days. An enzyme-linked immunosorbent assay (ELISA) kit was used to measure the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-10, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1.

Results

The chemical constituents of Nanocnide lobata mainly include volatile oils, coumarins, and lactones. UPLC–MS analysis revealed 39 main compounds in the Nanocnide lobata extract. Among them, ferulic acid, kaempferitrin, caffeic acid, and salicylic acid have been confirmed to exhibit anti-inflammatory and antioxidant activity related to the treatment of burns and scalds. HE staining revealed a gradual decrease in the number of inflammatory cells and healing of the wounds with increasing time after Nanocnide lobata extract administration. Compared with the model group, the petroleum ether extract-treated group showed significant differences in the levels of TNF-α (161.67±4.93, 106.33±3.21, 77.67±4.04 pg/mL) and IL-10 (291.77±4.93, 185.09±9.54, 141.33±1.53 pg/mL) on the 7^th, 14^th, and 21^st days; a significant difference in the content of TGF-β1 (75.68±3.06 pg/mL) on the 21^st day; and a significant difference in the level of VEGF (266.67±4.73, 311.33±10.50 pg/mL) on the 7^th and 14^th days respectively.

Conclusion

Petroleum ether Nanocnide lobata extract and the volatile oil compounds of Nanocnide lobata might be effective drugs in the treatment of burn and scald injuries, as they exhibited a protective effect on burns and scalds by reducing the expression of TNF-α, IL-10 and TGF-β1 and increasing the expression of VEGF. In addition, these compounds may also exert pharmacological effects that promote wound tissue repair, accelerate wound healing, and reduce scar tissue proliferation, inflammation and pain.

1. Introduction

Burn and scald injuries consist of damage to the skin or other body tissue caused by thermal radiation, which occurs when some or all of the cells of the skin or other tissue are damaged by hot liquids (scalds), solids (contact burns), or flames (flame burns) [1]. Other injuries to the skin or other body tissue caused by radiation, electrical current, friction, or exposure to chemicals are also considered burns [2]. Approximately 100 million people in the world suffer from burns of varying degrees every year, and the number of deaths caused by burns is second only to that caused by traffic accidents [3]. Severe burns and scalds cause both physical and mental harm, making it difficult for those affected to reintegrate into society [4]. In patients with large-scale burns and scalds, due to wound exudation and excessive water loss, the imbalance of body fluids and electrolytes is likely to cause fever, thereby reducing the physical fitness of the human body and affecting wound healing [5]. Additionally, long-term fever can easily lead to various complications and further endanger life. Burns and scalds can also cause serious damage to human skin, which may result in scars and disfigurement in severe cases [6].

Nanocnide lobata Wedd.N.pilosa Migo (snow medicine) is mainly distributed in shady and humid places in Chongqing, Sichuan, Hubei, Guangdong, Guangxi, Guizhou, Yunnan and other places in China. The family name of Nanocnide lobata is Urticaceae which less reports on the chemical components of it. It has been reported that the possible types of chemical components in Nanocnide lobata are organic acids, polysaccharides and glycosides, steroids or triterpenoids, flavonoids, coumarins, lactones, and volatile oils [7]. Nanocnide lobata is a traditional Chinese medicine (TCM) that is usually used to treat lung heat and cough, scrofula, hemoptysis, burns and scalds, carbuncles, bruises, snakebites, and traumatic bleeding [7,8]. Nanocnide lobata is commonly used to treat burns and scalds in Chinese folk medicine. However, there has been little modern clinical research on the pharmacology and efficacy of Nanocnide lobata in the treatment of burn and scald injuries [7,8]. In addition, there have been no systematic studies on the active fraction or chemical constituents of Nanocnide lobata in the treatment of burn and scald injuries.

The chemical composition of medicinal materials is the basis for their pharmacology and clinical efficacy. In the present experiment, petroleum ether, ethyl acetate and n-butanol were used to extract different active fractions of Nanocnide lobata. Chemical identification methods were utilized to investigate the chemical composition of each extracted fraction of Nanocnide lobata and provide a chemical foundation for further mechanistic research. In addition, active components in the treatment of burn and scald injuries were clarified.

2. Materials and methods

2.1 Materials

The original herbarium of Nanocnide lobata was kept in the 407 Natural medicinal chemistry Laboratory of the Scientific Research Center of Chongqing Three Gorges Medical College (No.20210503) in Wanzhou District, Chongqing, China. Specimens were gathered in April 2021 and identified as the aerial part of Nanocnide lobata Wedd., a plant of the Urticaceae family, by Professor Yi, School of Pharmacy, Chongqing Three Gorges Medical College. Approximately 760 g of dried Nanocnide lobata herbs was weighed and pulverized into primary powder. Then, 12 L (4 L×3) of 95% ethanol was added; the mixture was soaked for 48 h and then filtered with a 200-mesh filter cloth to obtain the filtrate. The filtrate was concentrated under reduced pressure to obtain 208.32 g of total extract at 40°C and 0.1 MPa. Nanocnide lobata extraction was performed with ethanol and an appropriate amount of water using Han’s method [9], with petroleum ether using Hacıbekiroğlu’s method [10], with ethyl acetate using Afsar’s method [11] and with n-butanol using Stoffers’s method [12] 3 times at a ratio of 1:1. Finally, the mixture was concentrated under reduced pressure to obtain 15.83 g of petroleum ether extract, 2.54 g of ethyl acetate ester extract, and 13.58 g of n-butanol extract. Then, the petroleum ether extract, ethyl acetate ester extract, and n-butanol extract were each mixed with white Vaseline at a proportion of 85% white Vaseline and 15% extract to obtain ointments of suitable consistency for the further screening of active fractions.

2.2 Reagents

Anhydrous ethanol, 95% ethanol, petroleum ether (60~90°C), n-butanol, ethyl acetate, ammonia water, potassium hydroxide, hydrochloric acid, sodium hydroxide, sulfuric acid, glacial acetic acid, acetic anhydride, chloroform, trichloromethane ferric chloride, sodium chloride, gelatin, vanillin, ninhydrin, copper sulfate, α-naphthol, copper sulfate, potassium sodium tartrate, bromophenol blue, bromocresol green, aluminum trichloride, magnesium powder, hydrogen oxide, boric acid, magnesium acetate, hydroxylamine hydrochloride, 3,5-dinitrobenzoic acid, 2,4,6-trinitrophenol, pyridine, sodium nitroferricyanide, iodine, potassium iodide, bismuth subnitrate, sodium silicotungstate, phosphomolybdic acid, potassium ferricyanide, 1% pentobarbital sodium, 4% paraformaldehyde universal tissue fixative, white petrolatum, “Jing wan hong” ointment (positive drug) and other reagents used to prepare chemical solutions were all analytically pure, and the water was purified water. Enzyme-linked immunosorbent assay (ELISA) test kits, including a mouse vascular endothelial growth factor (VEGF) ELISA kit, mouse transforming growth factor β 1 (TGF-β1) ELISA detection kit, mouse tumor necrosis factor α (TNF-α) ELISA test kit, and mouse interleukin-10 (IL-10) ELISA test kit, were purchased from Shanghai Future Industry Co., Ltd., and used for further study.

2.3 Experimental animals

Specific pathogen-free (SPF) female mice (weighing 20–30 g) supplied by Hunan Slike Jingda Laboratory Animal Co., Ltd., were used in the experiment. They were kept in separate cages with 10 mice per cage in a clean-grade animal laboratory at constant temperature of 25±10°C with a relative humidity of 40% to 70%. The breeding environment just kept at light-dark cycle was 12 h. Adapt to feeding for consecutive 3 days, fasting 12 h before the experiment, and drinking water freely.

2.4 Instruments

The following equipment was used in this study: vertical blast drying oven (Shanghai Jinwen Instrument Co., Ltd.); RE-5298 rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); HLD-10002 electronic balance (Hangzhou Youheng Weighing Equipment Co., Ltd.); KH2200DB CNC Ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); HH-6 digital constant temperature water bath (Changzhou Yichen Instrument Manufacturing Co., Ltd.); SHZ-D (III) circulating water-type multipurpose vacuum pump (Henan Yuhua Instrument Co., Ltd.); BW-YLS-5Q desktop temperature control scald instrument (Shanghai Ruanlong Technology Development Co., Ltd.); HH-6 digital constant temperature water bath (Changzhou Yichen Instrument Manufacturing Co., Ltd.); DNM-9606 enzyme label analyzer (American Boten Instrument Co., Ltd.); 80–2 desktop centrifuge (Changzhou Jintan Liangyou Instrument Co., Ltd.); KD-BM biological tissue embedding machine (Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.); LEICA RM2235 slicer (Beijing Haonuo Technology Co., Ltd.); ultra-performance liquid chromatography (UPLC)–mass spectroscopy (MS) system (UPLC, Vanquish; MS, HFX, Thermo Scientific Co., Ltd.); Q Exactive HFX Hybrid Quadrupole Orbitrap mass spectrometer equipped with a heated electron spray ionization (ESI) source (Thermo Fisher Scientific Co., Ltd.); Q Exactive using Xcalibur 4.1 (Thermo Scientific Co., Ltd.); Progenesis QI software (Waters Corporation, Milford, USA); and Milli-Q water purification system (Millipore, Bedford, MA, USA).

2.5 Preliminary chemical identification tests

Preliminary chemical identification tests were carried out by a variety of color or precipitation reactions using indicator and chromogenic agents. Finally, the chemical composition of the Nanocnide lobata extracts were identified.

2.6 Extraction of bioactive ingredients from Nanocnide lobata

2.6.1 Preparation of samples

Nanocnide lobata powder was subjected to extraction by maceration with 95% ethanol overnight at room temperature for 3 consecutive days to yield the sample. Approximately 1 ml of the sample was added to 2 times the volume of the methanol-acetonitrile extraction solution (1:1, v/v), vortexed for 60 s, and sonicated for 30 min. After centrifugation (20 min, 12000 rpm, 4°C), the supernatant was transferred to a clean plastic microtube. The sample was incubated for 1 h at -20°C and then centrifuged at 12000 g at 4°C for 10 min to remove the protein. The mixture was then centrifuged for 10 min (12000 g, 4°C), and the supernatant was dried in a vacuum centrifuge. Subsequently, the sample was redissolved in 100 μl of 30% methyl cyanide (vol/vol) and transferred to an insert-equipped vial for analysis.

2.6.2 UPLC‒MS analyses

Analysis of the bioactive compounds of Nanocnide lobata was carried out using a UPLC‒MS system coupled to a Q Exactive HFX Hybrid Quadrupole Orbitrap mass spectrometer equipped with a heated ESI source utilizing the full-ms-ddMS2 MS acquisition method. The analytical conditions were as follows: UPLC: column, Waters HSS T3 (100×2.1 mm, 1.8 μm); column temperature, 40°C; mobile phase: 0.1% formic acid aqueous solution as phase A and 0.1% formic acid acetonitrile as phase B; flow rate, 0.3 mL/min; injection volume, 2 μL; solvent system, water (0.1% acetic acid): acetonitrile (0.1% acetic acid); and gradient program, 0 min, 0% phase B; 1 min, 0% phase B; 9 min, 95% phase B; 13 min, 95% phase B; 13 min, 0% phase B; 17 min, 0% phase B. The ESI source parameters were set as follows: spray voltage, -2.8 kV/3.0 kV; sheath gas pressure, 40 arb; aux gas pressure, 10 arb; sweep gas pressure, 0 arb; capillary temperature, 320°C; and aux gas heater temperature, 350°C. The raw MS data were acquired on the Q Exactive using Xcalibur 4.1 and processed using Progenesis QI.

2.7 Pharmaceutical trial validation

2.7.1 Establishment of the scald/burn model and treatment of mice

All mice were randomly divided into the following 6 groups, with 10 mice in each group: the petroleum extract-treated group; the ethyl acetate extract-treated group; the n-butanol extract-treated group; the positive drug group; the model group; and the control group. The scald/burn model was established following Stevenson’s method [13]. After mice in each group were anesthetized by an intraperitoneal injection of 1% pentobarbital sodium (80 mg/kg), fur across approximately 16 cm² (4 cm× 4 cm) of the mouse back was removed with a depilator, and the small amount of remaining fur was removed with depilatory cream. A copper metal rod with a diameter of 1 cm was heated to 95°C and placed on the depilated area for 15 s, resulting in a shallow second-degree scald, with a wound size of approximately 150–200 mm². Twenty-four hours after modeling, mice were treated topically with 0.1 g of the corresponding ointment on the wound every day in each group; administrated with 0.1 g of vaseline in the control group, administrated with 0.1 g of “Jing wan hong” ointment in the positive drug group, and were not treated in model group. The treatment was administered once a day for 21 successive days (Table 1). All mice were humanely sacrificed by the inhalation of CO₂ at a gradually increasing rate of 30–70% of the chamber volume/min upon meeting the following criteria: >20% weight loss; dyspnea; and dramatic drop in body temperature. The absence of movement and breathing, as well as cardiac arrest and pupil dilation for 5 min, were used to confirm death.

Table 1. Animal grouping and treatment.

Grouping	Dose of drug	Treated time
PE group	0.1 g petroleum extract	consecutive 21 days
EA group	0.1 g ethyl acetate extract	consecutive 21 days
NB group	0.1 g n-butanol extract	consecutive 21 days
C group	0.1 g vaseline	consecutive 21 days
M group	no drug administrated	consecutive 21 days
PD group	0.1 g “Jing wan hong” ointment	consecutive 21 days

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Note: PE group presents petroleum extract treated group, EA group presents ethyl acetate extract treated group, NB group presents n-butanol extract treated group, C group presents control group, M group presents model group, PD group presents positive drug group.

2.7.2 Measurement of wound healing rate

The drug was administered at the same time every day, and characteristics of the wound, including color, secretions, hardness and swelling, were observed and recorded. Photos were taken on the 1^st, 5^th, 8^th, 12^th, 15^th, 18^th and 21^st days after modeling. Then, ImageJ software was used to calculate the wound area and the rate of wound contracture as a percentage according to the following formula: wound contracture percentage on a certain day = wound area on a certain day/initial wound area × 100%.

2.7.3 Histopathological examination

On the 7^th, 14^th, and 21^st days after modeling, mice in each group were anesthetized by an intraperitoneal injection of 1% pentobarbital sodium (80 mg/kg). After stripping the wound to the muscle layer, the mice were sacrificed. Take approximately 1 mL of blood from the orbit of mice, deposit for 20 min, then centrifuge at 3000 rpm for 5 min, and take the upper serum. The upper serum layer was removed, placed in a clean centrifuge tube, and stored at -80°C for later use. Wound tissue samples were fixed with 4% paraformaldehyde fixing solution. Paraffin sections were created, and the sections were stained with hematoxylin-eosin (HE). After staining, the samples were examined for sweat glands, inflammatory cell infiltration, fibrous tissue hyperplasia, tissue deformation, necrosis and calcification. The histopathological evaluation score was determined according to the extent of the burn and scald injuries with the following grading system: 0 points for no or minimal pathological change, 1 point for mild condition, 2 points for moderate condition, 3 points for severe condition, and 4 points for extremely severe condition.

2.7.4 ELISA

Mouse blood samples were removed from storage, allowed to naturally coagulate at room temperature and then centrifuged at 3000 rpm for 20 min; then, the cell supernatant was collected using sterile EP tubes. Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-10, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1 according to the manufacturer’s instructions.

2.8 Data analysis

The wound healing area data were statistically analyzed using Excel 2010 and SPSS 20.0, and experimental data from each group are expressed as the mean ± standard deviation ( $\bar{x} \pm s$ ). P <0.05 and P <0.01 were considered statistically significant.

2.9 Ethics statement

Animal treatment and maintenance procedures were performed strictly in accordance with the Principle of Laboratory Animal Care and approved by the Animal Research Committee of Chongqing Three Gorges Medical College, Chongqing, China (License No. 2020–007).

3. Results

3.1 Chemical composition categories for each extraction fraction

The fraction of Nanocnide lobata obtained by petroleum ether extraction may contain volatile oils or grease, steroids, and triterpenoids (Table 2); the fraction obtained by ethyl acetate extraction may contain coumarins, lactones, and phenolic tannins (Table 3); the fraction obtained by n-butanol extraction may contain fragrance legumes, lactones, phenolic tannins, steroids and triterpenoids (Table 4); and the fraction obtained by water extraction may contain reducing sugars, polysaccharides, glycosides, phenolic tannins, amino acids, polypeptides, proteins, and organic acid class compounds (Table 5).

Table 2. Identification test of chemical composition of petroleum ether extraction part on Nanocnide lobata.

Chemical composition	Name of experiment	Positive reaction index	Result	Conclusion
Volatile oil or grease	Oil spot test Phosphomolybdic acid test	The oil spot can volatilize without leaving a trace at room temperature, indicating that there is volatile oil. The oil spots do not disappear, indicating that there are oils and fats.	Oil spots do not disappear	√
		Yellow-green background with spots in blue	Spots are blue	√
	Vanillin-sulfuric acid experiment	Spots are red, blue, purple, etc.	Spots are green	×
Steroids or triterpenoids	Chloroform-concentrated sulfuric acid test	The chloroform layer is red or cyan, and the sulfuric acid layer has green fluorescence when observed under UV light	The chloroform layer is red, and the sulfuric acid layer has green fluorescence	√
Steroids or triterpenoids	Acetic anhydride-concentrated sulfuric acid test	The color of the reaction solution changes from yellow→red→purple→blue→dirty green	The color of the reaction solution changes from yellow to red	√

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Note: "√" indicates positive reaction, "×" indicates negative reaction.

The experimental results show that the petroleum ether extraction part of Nanocnide lobata may contain volatile oil or grease, steroids or triterpenoids.

Table 3. Identification test of chemical composition of ethyl acetate extraction part on Nanocnide lobata.

Chemical composition	Name of experiment	Positive reaction index	Result	Conclusion
Flavonoids	Hydrochloric acid-magnesium powder reaction	The reaction solution or the resulting foam is red to purple	The foam is white	×
	Aluminum trichloride reaction	Visible yellow or yellow-green fluorescence when viewed under UV light	Spots are green	√
	Ammonia fumigation test	Spots are yellow fluorescent under UV light	Spots are green	×
Coumarin or lactones	Fluorescence experiment	The spots were blue fluorescent when inspected under UV light, and the spots changed from fluorescent color to yellow-green after spraying 1% potassium hydroxide reagent	Spots are yellow-green	√
Coumarin or lactones	Iron hydroxamate reaction	The reaction solution appears orange-red or purple	The reaction solution appears orange-red	√
Phenolic tannins	Ferric chloride reaction	Spots are green, blue-green, dark green, bluish-purple	Spots are blue	√
Phenolic tannins	Gelatin test Vanillin hydrochloric acid reaction	Precipitation is produced Spots appear red to varying degrees	No precipitation is formed Spots are green	× ×
Anthraquinones	lye test	The reaction solution is red, add hydrogen peroxide to heat, the red does not disappear, acidify with hydrochloric acid, the red disappears	The reaction solution has no color change	×
Anthraquinones	Boric acid solution test	Spots are orange-yellow or red and fluoresce when viewed under UV light	No change in spots, no fluorescent color	×
Alkaloids	Silicotungstic acid test	Light yellow or gray white precipitates fromed	No precipitate was formed, the solution was yellow	×
	Bismuth potassium iodide test	A yellow or orange-red precipitate formed	No precipitate formed, the solution was brown	×
	Potassium iodide test	A brown precipitate formed	No precipitate was formed, the solution was yellow-brown	×

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Note: "√" indicates positive reaction, "×" indicates negative reaction.

The experimental results show that the ethyl acetate extraction part of Nanocnide lobata may contain coumarin or lactones and phenolic tannins.

Table 4. Identification test of chemical composition of n-butanol extraction part on Nanocnide lobata.

Chemical composition	Name of experiment	Positive reaction index	Result	Conclusion
Flavonoids	Hydrochloric acid magnesium powder test	The reaction solution or the generated foam shows purplish red	White foam formed	×
	Aluminum trichloride reaction	Visible yellow or yellow-green fluorescence when viewed under UV light	No visible color change	×
	Ammonia fumigation test	Spots are yellow fluorescent under UV light	No visible color change	×
Coumarin or lactones	Fluorescence experiment	The spots are blue fluorescent when inspected under UV light, and the fluorescent color of the spots changed to yellow-green after spraying with 1% potassium hydroxide reagent	Spots are yellow-green	√
Coumarin or lactones	Iron hydroxamate reaction	The reaction solution is orange-red or purple-red	The reaction solution is orange-red	√
Anthraquinones	lye test	The reaction solution is red, add hydrogen peroxide to heat, the red does not disappear, acidify with hydrochloric acid, the red disappears	No visible color change	×
Anthraquinones	Boric acid solution test	Spots are orange-yellow or red and fluoresce when viewed under UV light	No change in spots, no fluorescent color	×
Phenolic tannins	Gelatin test	Precipitation formed	No precipitation formed	×
	Vanillin hydrochloric acid reaction	Spots appear red to varying degrees	Spots are colorless	×
	Ferric chloride reaction	Spots are green, blue-green, dark green, blue-purple	Spots are yellow-green	√
Steroids or triterpenoids	Chloroform-concentrated sulfuric acid test	The chloroform layer is red or cyan, and the sulfuric acid layer has green fluorescence when observed under UV light	The chloroform layer is red, and the sulfuric acid layer has green fluorescence	√
Steroids or triterpenoids	Acetic anhydride-concentrated sulfuric acid test	The color of the reaction solution changes from yellow→red→purple→blue→dirty green	The color of the reaction solution changes from yellow to red	√
Alkaloids	Silicotungstic acid test	A pale yellow or off-white precipitate formed	No precipitate was formed, the solution was yellow	×
	Bismuth potassium iodide test	A yellow or orange-red precipitate formed	No precipitate formed, the solution was brown	×
	Potassium iodide test	A brown precipitate formed	No precipitate was formed, the solution was yellow-brown	×

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Note: "√" indicates positive reaction, "×" indicates negative reaction.

The experimental results show that the n-butanol extraction part of Nanocnide lobata may contain coumarin or lactones, phenolic tannins, steroids or triterpenoids.

Table 5. Identification test of chemical composition of water extraction part on Nanocnide lobata.

Chemical composition	Name of experiment	Positive reaction index	Result	Conclusion
Reducing sugars, polysaccharides and glycosides	Molich reaction	A red halo appeared between the two liquid surfaces	A red circle appears	√
Reducing sugars, polysaccharides and glycosides	Fehling reaction	Precipitation changes from light blue→brown→brick red	A brick red precipitate is formed	√
Saponins	foam test	Foam appears after continuous shaking and does not disappear for a long time	A small amount of foam, which disappears after a short period of time	×
Saponins	Acetic anhydride-concentrated sulfuric acid test	The color of the reaction solution is from yellow→red→purple→blue→green	No significant changes	×
Phenolic tannins	Vanillin hydrochloric acid reaction	Spots are red	Spots are colorless	×
	Ferric chloride reaction	Green, dark green, bluish black or dark purple	Blue-black	√
	Gelatin test	A white precipitate is formed	No precipitation is formed	×
Amino acids, peptides and proteins	Ninhydrin test	Blue-purple or bright yellow	No color reaction	×
	Biuret test	Purple or bluish-purple	No color reaction	×
	Heating precipitation test	Heating and boiling → turbid precipitation	Precipitation is formed	√
Organic acids	Bromocresol green test	Spots are yellow on a blue background	yellow	√
	Bromophenol blue test	Spots are yellow on a blue background	yellow	√
	PH paper test	The color of the test paper is below PH7	PH between 6~7	√

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Note: "√" indicates positive reaction, "×" indicates negative reaction.

The experimental results show that the water extraction part of Nanocnide lobata may contain reducing sugars, polysaccharides and glycosides, phenolic tannins, amino acids, polypeptides and proteins, organic acid compounds.

3.2 UPLC‒MS results

Typical total ion chromatograms of the Nanocnide lobata samples in positive ion mode (Fig 1A) and negative ion mode (Fig 1B) are illustrated. A total of 39 compounds, including D-proline, raffinose, guanine, uridine, nicotinamide, pantothenic acid, protocatechuic acid, gentisic acid, kynurenic acid, esculin, ferulic acid, vicenin II, corymboside, isoschaftoside, caffeic acid, esculetin, cynaroside, isoorientin, N-acetyl-leucine, vitexin rhamnoside, isovitexin, isovitexin 2’’-O-arabinoside, kaempferitrin, vitexin, isoscoparin-2’’-beta-D-glucopyranoside, 4-hydroxycinnamic acid, syringaldehyde, scopoletin, salicylic acid, 2-indolecarboxylic acid, acuminoside, (+)-abscisic acid, 3-cresotinic acid, cumic alcohol, ethyl caffeate, diosmetin, skimmianine, 2-(3-methoxy-4-hydroxyphenyl)-5-(3,4-dimethoxyphenyl)-3,4-dimethyltetrahydrofur and usniacin, were identified or tentatively characterized. UPLC‒MS data, such as retention times, chemical formulas, compounds and main fragment ions, are summarized in Table 6. The above spectrum-effect relationship results illustrated that peaks P1 to P39 (S1 Table) were compounds in Nanocnide lobata potentially involved in the treatment of burn and scald injuries.

Table 6. The typical total ion chromatogram of Nanocnide lobata sample in positive ion mode and negative ion mode respectively.

NO.	Retention time (min)	Compound	Formula	[M+H,M+H-H₂O]	[M-H,M+FA-H]
1	0.8115	D-proline	C₅H₉NO₂	116.0707	-
2	0.8260	Raffinose	C₁₈H₃₂O	-	539.1384
3	0.9802	Guanine	C₅H₅N₅O	152.0564
4	1.044	Uridine	C₉H₁₂N₂O₆	-	243.0629
5	1.362	nicotinamide	C₆H₆N₂O	123.0552	-
6	2.799	Pantothenic acid	C₉H₁₇NO₅	220.1175	-
7	3.546	Protocatechuic acid	C₇H₆O₄	-	153.0198
8	3.905	Gentisic acid	C₇H₆O₄	-	153.0198
9	4.161	Kynurenic acid	C₁₀H₇NO₃	190.0494	-
10	4.230	Esculin	C₁₅H₁₆O₉	-	385.0788
11	4.258	ferulic acid	C₁₀H₁₀O₄	-	193.0512
12	4.533	Vicenin II	C₂₇H₃₀O₁₅	-	593.1537
13	4.712	Corymboside	C₂₆H₂₈O₁₄	565.1542	-
14	4.719	Isoschaftoside	C₂₆H₂₈O₁₄	-	563.1440
15	4.737	caffeic acid	C₉H₈O₄	-	179.0357
16	4.746	Esculetin	C₉H₆O₄	-	177.0200
17	4.792	Cynaroside	C₂₁H₂₀O₁₁	-	447.0944
18	4.804	Isoorientin	C₂₁H₂₀O₁₁	449.1070	-
19	4.879	N-Acetyl-leucine	C₈H₁₅NO₃	-	172.0986
20	4.964	Vitexin rhamnoside	C₂₇H₃₀O₁₄	-	577.1590
21	4.966	Isovitexin	C₂₁H₂₀O₁₀	433.1121	-
22	4.966	Isovitexin 2’’-O-arabinoside	C₂₆H₂₈O₁₄	565.1541	-
23	5.078	Kaempferitrin	C₂₇H₃₀O₁₄	-	577.1590
24	5.088	Vitexin	C₂₁H₂₀O₁₀	-	431.0998
25	5.182	Isoscoparin-2’’-Beta-D-glucopyranoside	C₂₈H₃₂O₁₆	-	605.1532
26	5.392	4-Hydroxycinnamic acid	C₉H₈O₃	-	163.0407
27	5.467	Syringaldehyde	C₉H₁₀O₄	-	181.0514
28	5.513	scopoletin	C₁₀H₈O₄	-	191.0357
29	5.617	Salicylic acid	C₇H₆O₃	-	137.0247
30	5.714	2-Indolecarboxylic acid	C₉H₇NO₂	-	160.0410
31	6.065	Acuminoside	C₂₁H₃₆O₁₀	-	447.2248
32	6.066	(+)-Abscisic acid	C₁₅H₂₀O₄	-	263.1293
33	6.289	3-Cresotinic acid	C₈H₈O₃	-	151.0405
34	6.579	Cumic alcohol	C₁₀H₁₄O	133.1010	-
35	6.597	Ethyl caffeate	C₁₁H₁₂O₄	-	207.0670
36	6.856	Diosmetin	C₁₆H₁₂O₆	301.0699	-
37	7.157	Skimmianine	C₁₄H₁₃NO₄	260.0912	-
38	8.535	2-(3-Methoxy-4-hydroxyphenyl)-5-(3,4-dimethoxyphenyl)-3,4-dimethyltetrahydrofuran	C₂₁H₂₆O₅	341.1738	-
39	10.31	Usniacin	C₁₈H₁₆O₇	-	343.0835

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3.3 Observation of scald/burn wounds

By arranging the photos of wounds in each group of mice at different times with the same scale adjustment, the wound recovery in each group could be visually observed. The results are shown in Fig 2. From the 2^nd day to the 8^th day, the wounds in each group showed blisters, with thin, brownish-yellow, soft crusts. The ointments applied in each group adhered well to the wounds. From the beginning of the 2^nd day, the wounds in the petroleum ether extract-treated group became hard, and the wounds in the control group were wet. Due to the darker color of the ointment, the wounds in the petroleum ether extract-treated group were darker. From the 8^th day to the 12^th day, slight effusion was observed in the control and model groups. From the 12^th day onward, the wounds in each group showed an obvious decrease in the degree of wound contracture, except the wounds in the model group remained larger than those in the other groups. After the 15^th day, the wounds in each group had healed well, with no bleeding, and were brownish in color. The wound area was significantly smaller than that in the other groups. On the 21^st day, the treatment effect in the petroleum ether extract-treated group was the most significant; the wound had basically healed, and the healed surface was smooth.

3.4 Wound healing rate results

After the 8^th day, the wound area as a percentage of the initial area was the smallest in the positive drug group, followed by the petroleum ether extract-treated group and n-butanol extract-treated group, with a significant difference in the petroleum ether extract-treated group and positive drug group compared with the model group (P<0.05). After the 12^th day, a significant difference was observed in the petroleum ether extract-treated group and the positive drug group compared with the model group (P<0.01). From the 18^th day, the smallest wound area as a percentage was observed in the petroleum ether extract-treated group, followed by the control group and n-butanol extract-treated group (S2 Table). The percent remaining wound area in the ethyl acetate extract-treated group was significantly different from that in the control group. The percent remaining wound area was similar in the ethyl acetate extract-treated group and the model group and lowest in the petroleum ether extract-treated group (Table 7).

Table 7. Change in percent contracture rate of wound ( $\bar{x} \pm s$ ).

Time (d)	wound contracture rate (%)
Time (d)	PE group	EA group	NB group	C group	M group	PD group
Day 1	100±0.0	100±0.0	100±0.0	100±0.0	100±0.0	100±0.0
Day 5	83.37±11	99.07±11	84.02±29	96.63±24	92.02±25	73.28±17
Day 8	70.75±17^*	75.23±23	64.86±34	95.21±6^*	78.87±26	50.98±4^*
Day 12	20.97±7^**	53.72±20	37.23±21	58.82±18	59.15±18	18.29±10^**
Day 15	15.44±6^**	33.13±13^*	36.27±16	34.47±13	47.32±15	15.21±7^**
Day 18	3.79±4^**	11.61±8	20.34±24	5.47±17	20.85±16	7.32±4^**
Day 21	0.97±1^**	4.21±7	4.52±6	1.64±6	11.83±8	1.48±2^**

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Note: n = 10

*P<0.05

**P<0.01.

PE group presents petroleum extract treated group (0.1 g petroleum extract each day), EA group presents ethyl acetate extract treated group (0.1 g ethyl acetate extract each day), NB group presents n-butanol extract treated group (0.1 g n-butanol extract each day), C group presents control group (0.1 g vaseline each day), M group presents model group (no drug administrated), PD group presents positive drug group (0.1 g “Jing wan hong” ointment each day).

3.5 HE staining results

On the 7^th day after modeling, no significant effect on wound healing was observed in the petroleum ether extract-treated group (Fig 3A) or the n-butanol extract-treated group (Fig 3B). Observation of the pathological sections from the mice in the petroleum ether extract-treated group (Figs 4A and 5A) and the n-butanol extract-treated group (Figs 4B and 5B) showed a gradual decrease in the number of inflammatory cells with increasing time until the 14^th and 21^st days after modeling. It was also observed that the bleeding was well controlled, indicating that the wounds had basically healed. In addition, in the ethyl acetate extract-treated group (Figs 3C, 4C and 5C), most of the necrotic epidermal tissue sloughed off, epidermal migration was observed on the surface of the wound edge, increases in the numbers of fibroblasts and capillaries were observed, and there was still mild inflammatory cell infiltration in the dermis. The treatment effect was slightly better in the ethyl acetate extract-treated group than in the model group, but the wounds had still not fully healed. On the 7^th, 14^th, and 21^st days after modeling, no inflammatory cell aggregation was observed in pathological sections from the control group (Figs 3D, 4D and 5D). In the model group, large ulcers containing inflammatory cells, mild inflammation and moderate bleeding in the dermis were observed, with severe peeling and crusting of the epidermis, indicating a lack of wound healing (Figs 3E, 4E and 5E). Compared with those in the model group, wounds in the positive drug group showed mild epidermal hyperplasia and a large number of sweat glands generated in the dermis, indicating wound healing (Figs 3F, 4F and 5F). In addition, according to the score of evaluation standards of tissues, significant pathological changes were observed (Fig 6). Compared with the control group, the score significant increased in the model group (**P<0.01) while decreased in petroleum extract treated group (^##P<0.01), ethyl acetate extract group (^##P<0.05, ^##P <0.05) and n-butanol extract treated group (^##P<0.01) at day 7 (Fig 6A), day 14 (Fig 6B) and day 21 (Fig 6C) respectively. Taken together, the histopathological results were basically consistent with the results of overall observation.

Fig 4 — (A) petroleum ether extract-treated group, (B) n-butanol extract-treated group, (C) ethyl acetate extract-treated group, (D) control group, (E) model group, (F) positive drug group.

Fig 5 — (A) petroleum ether extract-treated group, (B) n-butanol extract-treated group, (C) ethyl acetate extract-treated group, (D) control group, (E) model group, (F) positive drug group.

Fig 6 — (A) day 7, (B) day 14, (C) day 21.

3.5 ELISA results

The serum levels of TNF-α and IL-10 on the 7^th, 14^th, and 21^st days were measured to investigate the effect of Nanocnide lobata on the inflammatory response. The serum level of TGF-β1 was measured to study the role of Nanocnide lobata in promoting wound healing, and the VEGF level was measured to investigate the effect of Nanocnide lobata on wound contracture (S3 Table). Compared with the model group, the petroleum ether extract-treated group showed significant differences in the serum levels of TNF-α (Table 8) and IL-10 (Table 9) on the 7^th, 14^th, and 21^st days. There was also a significant difference in the content of TGF-β1 in the petroleum ether extract-treated group on the 21^st day (Table 10) and in the content of VEGF in the petroleum ether extract-treated group on the 7^th and 14^th days (Table 11).

Table 8. Changes in the concentration of TNF-α in each group at different time points after injury ( $\bar{x} \pm s$ ).

Group	TNF-α (pg /mL) $\bar{x} \pm s$
Group	Day 7	Day 14	Day 21
PE group	161.67±4.93^**	106.33±3.21^*	77.67±4.04^**
EA group	194.57±5.86	156.38±2.08	98.65±2.08
NB group	191.69±6.11	148.31±4.16	99.01±1.00
C group	193.33±1.53	153.60±16.07	95.77±8.14
M group	199±1.02	153.39±15.27	103.33±2.87
PD group	157.67±1.53^**	103.03±4.25^*	78.01±2.19^**

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Note: n = 10

*P<0.05

**P<0.01.

Table 9. Changes in the concentration of IL-10 in each group at different time points after injury ( $\bar{x} \pm s$ ).

Group	IL-10 (pg /mL) $\bar{x} \pm s$
Group	Day 7	Day 14	Day 21
PE group	291.77±4.93^{* *}	185.09±9.54^**	141.33±1.53^{* *}
EA group	328.51±8.51	212.02±3.61	164.03±6.56
NB group	327.95±6.43	215.00±4.93	167.89±8.02
C group	323.33±1.53	215.33±5.51	173.23±2.01
M group	338.04±1.21	227.10±6.08	179.56±1.75
PD group	289.79±4.73^{* *}	175.69±2.52^**	146±2.66^{* *}

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Note: n = 10

*P<0.05

**P<0.01.

Table 10. Changes in the concentration of TGF-β1 in each group at different time points after injury ( $\bar{x} \pm s$ ).

Group	TGF-β1 (pg /mL) $\bar{x} \pm s$
Group	Day 7	Day 14	Day 21
PE group	100.01±4.58	97.67±4.16	75.68±3.06^*
EA group	113.02±13.11	116.34±6.81	113.12±7.21
NB group	111.33±6.81	118.23±13.11	118.97±2.52
C group	97.23±11.00	108.93±9.45	101.52±3.06
M group	117.15±12.11	114.69±11.72	115.01±5.03
PD group	88±4.58^*	108±4.58	91.77±3.01^*

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Note: n = 10

*P<0.05.

Table 11. Changes in the concentration of VEGF in each group at different time points after injury ( $\bar{x} \pm s$ ).

Group	VEGF (pg /mL) $\bar{x} \pm s$
Group	Day 7	Day 14	Day 21
PE group	266.67±4.73^*	311.33±10.50^*	270.33±5.13
EA group	228.00±3.01	264.33±7.02	281.36±8.62
NB group	238.33±3.51	269.67±6.11	280±8.02
C group	217.33±11.59	235.01±8.12	225.01±5.02
M group	217.67±14.64	239.33±17.6	247.11±14.03
PD group	315.67±9.5^**	322.67±2.08^*	320±5.58^*

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Note: n = 10

*P<0.05

**P<0.01.

4. Discussion

The healing burns and scalds is a natural physiological process that restores the function and integrity of damaged skin tissue [14]. The process is commonly divided into 4 overlapping stages, including coagulation, inflammation, neotissue formation and tissue remodeling. Cytokines and inflammatory mediators are involved in various processes of wound healing. The cells involved in wound healing mainly include keratinocytes, fibroblasts, endothelial cells, macrophages and platelets [15]. Cytokines include growth factors, tumor necrosis factors, interferons, colony-stimulating factors, chemokines, and interleukins [16]. The coagulation stage mainly involves platelets and fibrin, while the inflammatory stage mainly involves neutrophils, mononuclear macrophages, and lymphocytes, among others [17]. The proliferation stage mainly involves fibroblasts, endothelial cells, epithelial cells and collagen, and the remodeling stage mainly consists of collagen fiber contraction and scarring [18].

Western medicine and TCM have their own advantages and disadvantages in the treatment of burns and scalds [19]. Western medicine is well suited for treating patients with severe burns over large areas [20]. Because the onset of action is fast in Western medicine, it plays an irreplaceable role in rescuing critically burned patients. However, there are still some aspects of Western medicine that merit additional consideration, such as the long-term application of antibiotics and analgesics [21], which can lead to patients developing drug resistance and drug dependence. In TCM, anti-inflammatory, muscle-building, and heat- or toxin-removing drugs are often used and are the most commonly used drugs in the treatment of burn wounds in clinical practice [22]. Moreover, most Chinese medicines contain analgesic ingredients; thus, there is no need to use additional analgesics, which avoids the development of dependence on opioid analgesics [23].

The experimental results of this study showed that the fractions of Nanocnide lobata extracted using petroleum ether, ethyl acetate, n-butanol and water mainly contain volatile oils, coumarins, lactones, phenolic tannins, steroids, triterpenoids, reducing sugars, polysaccharides and glycosides, phenolic tannins, amino acids, polypeptides and proteins, and organic acid compounds. Similar to our results, it has been reported that volatile oils may have a beneficial effect on the healing of burns and scalds [24,25]; reducing sugars may also be beneficial for burns and scalds [26]. In addition, phenolic tannins and phenolic compounds have been reported to have a significant curative effect on burns and scalds, which is consistent with our results [27]. Furthermore, terpenoids are considered to have anti-inflammatory and antibacterial effects in the treatment of burns and scalds [28,29]. The combination of UPLC and MS can fully leverage their advantages, significantly improving the repeatability and reliability of quantitative analysis, as well as the accuracy of qualitative analysis. It has well adapted to the demand for automated and high-throughput analysis methods in modern drug research and has become one of the important methods for drug analysis (Table 6). Therefore, bioactive compounds in Nanocnide lobata were identified by UPLC‒MS. Among them, ferulic acid, kaempferitrin, caffeic acid, and salicylic acid have been confirmed to exhibit anti-inflammatory and antioxidant activity related to the treatment of burns and scalds. The anti-inflammatory effect of ferulic acid is mainly related to the levels of PPARγ and CAM and the NF-κB and p38 MAPK signaling pathways. Ferulic acid also plays an antifibrotic role via TGF-β/Smad and MMP/TIMP signaling [30]. A study on the anti-inflammatory activity of kaempferitrin showed that it inhibited leukocyte infiltration and exudation in mice with bradykinin-induced pleurisy [31]. Caffeic acid has been reported to exhibit anti-inflammatory, antioxidant and immunomodulatory properties by inhibiting NF-κB activation and transcriptional activity of the COX-2 gene in epithelial cells and iNOS gene expression and NO production in macrophage cell lines [32]. Salicylic acid plays a crucial role in defending against pathogenic agents and exerts anti-inflammatory effects through suppressing the transcription of cyclooxygenase genes [33]. In this mouse model of deep second-degree burns and scalds, macroscopic observation of wounds, calculation of the wound contracture and observation of pathological sections revealed the best therapeutic effect of Nanocnide lobata in the petroleum ether-treated group. The results of this study showed that secretions, redness and swelling of the burn and scald wounds resolved more quickly in the petroleum extract-treated group and n-butanol extract-treated group than in the model group (P < 0.05). Additionally, the wound healing rate was higher in the petroleum extract-treated group than in the ethyl acetate extract-treated group and n-butanol extract-treated group (P < 0.05), indicating a definite effect of Nanocnide lobata in the treatment of burns and scalds in mice. This drug may help to shorten the duration of clinical symptoms and promote rapid recovery from such wounds. In the clinical use of Nanocnide lobata for the treatment of burns and scalds, soaking with rapeseed oil is also applied, which is consistent with the active compounds (petroleum extract-treated group) identified in this study.

TNF-α and IL-10 are representative inflammatory factors involved in the inflammatory phase [34]. TNF-α is related to the initiation of early wound healing, while IL-10 can regulate the early inflammatory response to reduce stromal activity and avoid scar formation [35]. TGF-β1 can promote wound healing, but its overexpression can increase scar formation [36]. VEGF is one of the most potent angiogenic growth factors in skin. VEGF contributes to wound contracture, and while its underexpression can lead to wound healing, its overexpression may increase scar formation [37]. Several studies have shown that many important biological factors, including TNF-α, VEGF, and TGF-1, are involved in the process of burn wound repair [38,39]. The body is prompted to release proinflammatory cytokines such as TNF-α when the skin is injured and an infection develops. The cytokine eventually starts an inflammatory cascade reaction to eliminate necrotic cells and tissues [40]. However, individuals who have excessive inflammation may develop systemic inflammatory response syndrome (SIRS) and immunological dysfunction, which can be life-threatening [41]. Anti-inflammatory medications are therefore crucial for the healing of burn wounds. In the present study, significant differences in the serum levels of TNF-α and IL-10 were observed in the petroleum ether-treated group at the 7^th, 14^th, and 21^st days compared with the model group. There was also a significant difference in the content of TGF-β1 in the petroleum ether-treated group at the 21^st day compared with the model group and in the content of VEGF in the petroleum ether-treated group at the 7^th and 14^th days compared with the model group. An increase and then a decrease were observed in the levels of inflammatory indicators in the petroleum ether-treated group, with significant differences on the 7^th and 14^th days compared with the model group.

5. Conclusion

Taken together, the results of this study revealed that the main types of components in Nanocnide lobata include volatile oils, coumarins, and lactones. Among them, ferulic acid, kaempferitrin, caffeic acid, and salicylic acid may exhibit beneficial activities for the healing of burns and scalds. The ointment used in the petroleum ether treated group and the volatile oil compounds of Nanocnide lobata might be effective drugs in the treatment of burns and scalds, with protective pharmacological effects achieved by reducing the expression of TNF-α, IL-10 and TGF-β1 and increasing the expression of VEGF, in turn promoting wound tissue repair, accelerating wound healing, exerting anti-inflammatory and analgesic effects, and even reducing scar tissue proliferation.

Supporting information

S1 Table. The detailed information of components identified in Nanocnide lobata based on UPLC-MS.

(XLSX)

Click here for additional data file.^{(13.7KB, xlsx)}

S2 Table. The detailed information of contracture rate of wound.

(XLSX)

Click here for additional data file.^{(18.4KB, xlsx)}

S3 Table. The detailed information of ELISA on TNF-α, IL-10, TGF-β1, VEGF respectively.

(XLSX)

Click here for additional data file.^{(12.9KB, xlsx)}

Acknowledgments

All of the authors have developed research plans and participated in research design, manuscript development, editing, and completion of manuscripts. All authors contributed to manuscript revision, read and approved the submitted version.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by Chongqing Municipal Education Commission Science and Technology Research Project---“Research on the active ingredients of Nanocnide lobata (snow medicine) in the treatment of burns and scald”, NO. KJQN201802705; Chongqing Municipal Health and Family Planning Commission Traditional Chinese Medicine Science and Technology Project---“Pharmacodynamic observation and mechanism study of Nanocnide lobata (snow medicine) on burns and scald”, NO. ZY201702136; Scientific research and seedling breeding project of Chongqing Medical Biotechnology Association, cmba2022kyym-zkxmQ0003; and 2022 scientific research project of Chongqing Medical and Pharmaceutical College, ygz2022104 respectively.

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PLoS One. doi: 10.1371/journal.pone.0287147.r001

Decision Letter 0

Sairah Hafeez Kamran

14 Feb 2023

PONE-D-22-27583Investigating active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scaldPLOS ONE

Dear Dr. mao,

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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We look forward to receiving your revised manuscript.

Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

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2. Thank you for stating in your Funding Statement:

“This work was supported by Chongqing Municipal Education Commission Science and Technology Research Project---“Research on the active ingredients of Nanocnide lobata (snow medicine) in the treatment of burns and scald” (NO. KJQN201802705); Chongqing Municipal Health and Family Planning Commission Traditional Chinese Medicine Science and Technology Project---“Pharmacodynamic observation and mechanism study of Nanocnide lobata (snow medicine) on burns and scald” (NO. ZY201702136) respectively.”

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Additional Editor Comments (if provided):

Dear Authors

The manuscript requires scientific editing. The active plant extracts require quantitative analysis. The histopathological analysis also require detailed elucidation with proper marking in the figures of the prominent features

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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Reviewer #1: Yes

Reviewer #2: 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: Yes

Reviewer #2: No

**********

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: 1. There should be more investigation of chemical composition of different extracts that used in this Study and it is unacceptable to say 'may contains' that repeated for all extracts. You should use advanced method of analysis like GC- MS or HPLC or HPLC MS to be sure about the chemical constituents of the used extracts.

2. What is the time of plant collection?

3. In line 80: Using 15% of the plant extracts. What is your reference about that?

4. Resolution of picture needs to be improved.

5. Tables 5-9: Groups should be rewritten with clarified way.

Reviewer #2: Reviewer Comments:

1. Authors should rectify the grammar mistake throughout the manuscript.

2. Abstract:

Authors frequently used abbreviations in the whole abstract section.

Line#7: Confirm the “colorrections” spelling.

3. Introduction:

Line # 57 to 61, Please rewrite the whole sentences because the meaning of these is not clear.

4. Methodology section:

Line # 88 to 94, the authors should use a small letter throughout a sentence.

In lines # 95 to 97, the authors should use an ELISA test kit.

In line # 94, kindly check, whether the sentence is in the present tense or past tense.

In line # 100, authors should use the first full form then next time use abbreviations.

5. Result section:

In lines # 182 to 187, the authors should mention Table-1 with these lines.

In lines # 216 to 221, the authors should rectify the typographic and spelling mistakes.

6. Discussion section:

In lines # 254 to 256, the authors should complete the sentence (especially neotissue formation and tissue remodeling-----).

In lines # 256 to 259, rephrase the sentence.

In line # 274, explain the local burn wound. The authors should remove the word “local”.

In line # 282, rectify the “revelaed” spellings and check spelling throughout the manuscript.

In lines # 298 to 299, mention which active part.

Authors may relate their research work with existing literature.

7. Conclusion: the conclusion section should be concise.

**********

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Reviewer #1: No

Reviewer #2: Yes: Mehwish Mushtaq

**********

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Attachment

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PLoS One. 2023 Jun 13;18(6):e0287147. doi: 10.1371/journal.pone.0287147.r002

Author response to Decision Letter 0

31 Mar 2023

Review Comments to the Author

Dear editor and reviewer,

Thanks for your letter and for the reviewer and editor’ comments concerning our manuscript entitled "Investigating active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald" (Manuscript ID PONE-D-22-27583). We really appreciate Plos One for giving us an opportunity to revise our manuscript. The manuscript has been carefully and extensively revised according to the editor and the reviewers’ helpful comments. The changes have been highlighted in blue (reviewer 1#), red (reviewer 2#) and green (editor) of the revised manuscript respectively, all the co-authhave read and approved the revised manuscript.

And the responses were as follows:

Reviewer #1:

1. There should be more investigation of chemical composition of different extracts that used in this study and it is unacceptable to say 'may contains' that repeated for all extracts. You should use advanced method of analysis like GC- MS or HPLC or HPLC MS to be sure about the chemical constituents of the used extracts.

Response: Following your suggestion, UPLC-MS experiment has been carried out for clarify the chemical constituents of the used extracts. Please check the details of results in figure 1 and Table 5.

2.What is the time of plant collection?

Response: The time of plant collection is April of 2021 which has been added in the manuscript.

3.In line 80: Using 15% of the plant extracts. What is your reference about that?

Response: I’m so sorry for the mistake. The sentence of the meaning is “The above 3 parts are mixed evenly with the proportion of 85% white vaseline and 15% extractions (petroleum ether extraction, ethyl acetate ester extraction, and n-butanol extraction) respectively. For the purpose to obtain an ointment of suitable consistency for the screening experiment of active fractions for the further study.” which has been revised in line 81 to line 85.

4.Resolution of picture needs to be improved.

Response: Thanks for your careful work. The images of whole manuscript has been improved and re-uploaded as separate files.

5. Tables 5-9: Groups should be rewritten with clarified way.

Response: Thank you for your help. The groups have been rewritten with clarified and briefly way. Please check in the table captions part of the manuscript.

Reviewer #2: Reviewer Comments:

1.Authors should rectify the grammar mistake throughout the manuscript.

Response: Thank you for your kindly suggestion. The professor Wang of Chongqing Medical and Pharmaceutical College was invited to revised the manuscript. Please check the modified part which highlight in red.

2. Abstract:

Authors frequently used abbreviations in the whole abstract section.

Response: Due to the limitation of the magazine on the number of words in the abstract, the author used a lot of abbreviations in the abstract section. Please kindly be informed. Following your advice, the abbreviations was improved. Please check it in line 13 to line 17.

Line#7: Confirm the “colorrections” spelling.

Response: Thanks for your careful work. “colorrections” was revised to“color rections” . Please check it in line 7.

3. Introduction:

Line # 57 to 61, Please rewrite the whole sentences because the meaning of these is not clear.

Response: We really appreciated your careful work. Following your useful advice. The whole sentences has been revised. Please check it in line 61 to line 65.

4. Methodology section:

Line # 88 to 94, the authors should use a small letter throughout a sentence.

Response: The small letter was used throughout the sentence.

In lines # 95 to 97, the authors should use an ELISA test kit.

Response: The enzyme linked immunosorbent assay (ELISA) test kit was inserted in the manuscript.

In line # 94, kindly check, whether the sentence is in the present tense or past tense.

Response: Thanks for your careful work. The sentence is in past tense which had been revised.

In line # 100, authors should use the first full form then next time use abbreviations.

Response: Following your advice, the issues had been solved in the whole manuscript. Please check it in line 105.

5. Result section:

In lines # 182 to 187, the authors should mention Table-1 with these lines.

Response: Following your suggestion, the Tables with these lines were provided in the manuscript. Please check it in line 224 to line 230.

In lines # 216 to 221, the authors should rectify the typographic and spelling mistakes.

Response: Thanks for your careful work. The sentence lines 280 to 286 had been rectified one by one. Please check it in line 280 to line 286.

6. Discussion section:

In lines # 254 to 256, the authors should complete the sentence (especially neotissue formation and tissue remodeling-----).

Response: Thanks for your careful work. The sentence was revised which can be easily understood. Please check it in line 313 to line 315.

In lines # 256 to 259, rephrase the sentence.

Response: Following your advice, the sentence was rephrased. Please check it in line 315 to line 317.

In line # 274, explain the local burn wound. The authors should remove the word “local”.

Response: The word “local” was removed.

In line # 282, rectify the “revelaed” spellings and check spelling throughout the manuscript.

Response: The word “revelaed” spellings was rectified to revealed. Please check it in line 341.

In lines # 298 to 299, mention which active part.

Response: The active part (petroleum ether extraction) had been mentioned. Please check it in line 359.

Authors may relate their research work with existing literature.

Response: Thanks for your kindly help. Please check the related research work (line 367 to line 375) with refs (line 515 to line 528).

7.Conclusion: the conclusion section should be concise.

Response: Following your advice, the conclusion section was rewrite which is concise and brief. Please check it in line 384 to line 392.

*Corresponding author: Jingxin Mao, Ph. D

College of Pharmaceutical Sciences, Southwest University, 400715, Chongqing, China.

School of Pharmacy, Chongqing Medical and Pharmaceutical College, 401331, Chongqing, China.

Email: mmm518@163.com or maomao1985@email.swu.edu.cn

Tel: +86-13752922258

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(19KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0287147.r003

Decision Letter 1

Sairah Hafeez Kamran

24 Apr 2023

PONE-D-22-27583R1Investigating active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scaldPLOS ONE

Dear Dr. mao,

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

ACADEMIC EDITOR:

Dear Authors

As per the reviewers’ comments all amendments have been addressed. The results are well established, but the entire manuscript requires improvements and clarity in English and Scientific language.

Abstract: The section is lacking appropriate scientific language for e.g.The authors have stated “Objective: To analyze the initial chemical constituents of Nanocnide lobata and figure out the effect and active fraction in the treatment of burn and scald”.

What do the authors mean by initial chemical constituents (are they analyzing primary metabolites?) and “figure out the effect” which effect?

I believe that the Objective statement may be clearer and more understandable and what I perceive from the study, more likely may be stated as “To analyze the most effective fraction of Nanocnide lobata in the treatment of burn and scald and determine its bioactive constituents”

In the methods authors have stated paper assays and test tube assays. I believe that these are not scientific names of the assays, so please add clarity. “Petroleum extraction group” doesn’t depict the proper meaning. “Petroleum extract treated group” is one suggestion or authors may think of a better scientific depiction. What treatment was provided to the control group, model group and positive model group.

Which fraction of Nanocnide lobata was subjected to UPLC-MS analysis?

Conclusion shall also be restructured. “This study preliminarily (what do the authors mean by preliminary) identified the type of the chemical compounds and the effect of Nanocnide lobata in treatment of burn and scald, which provides the basically foundation for further research on its medicinal value”. This statement doesn’t provide a conclusion of the study.

The entire abstract section requires English and scientific language recheck. Many parts are vague and some are mentioned above.

Similarly Introduction section also requires a thorough recheck

Line 45: "Long-term fever is prone to various complications and life-threatening" incomplete sentence

Line 53-54: Please recheck

Line 56-57. If clinical efficacy has been proven then what are the authors trying to address in the study?

Please recheck Line 65-67.

Line 77 -85 are still not clear. The authors have mixed all the fractions and prepared an ointment, then how the authors are creating separate groups of animals with labels “Petroleum extraction group”……..

The preparation of ointment may be moved to methods section. Line 134, 137, 140, 143 headings may be restructured. Also provide references for the phytochemical tests performed for every fraction.

Line 148: “Preparation of samples” heading may be more appropriate.

Section 3.3 also requires restructuring.

Line 231 and 301, reconsider the headings

Discussion: Lines 328, 331-337, 342-348, 360-362, 378-385 require special attention as the scientific and English language are not correct.

Have the authors identified any bioactive compounds from the UPLC-MS, effective in the treatment of burns and scalds. They may mention them in the discussion section.

Conclusion section also requires restructuring.

Please recheck the table and figure numbers within text are mentioned appropriately.

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

Please submit your revised manuscript by Jun 08 2023 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'.

We look forward to receiving your revised manuscript.

Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

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

PLoS One. 2023 Jun 13;18(6):e0287147. doi: 10.1371/journal.pone.0287147.r004

Author response to Decision Letter 1

10 May 2023

As per the reviewers’ comments all amendments have been addressed. The results are well established, but the entire manuscript requires improvements and clarity in English and Scientific language.

Response: Following your suggestion, the entire manuscript has been revised by American Journal Experts (https://www.aje.cn/). Please find the Editing Certificate uploaded as an attachment.

Response: Thank you for your suggestions. The sentence has been revised to “To identify the most effective fraction of Nanocnide lobata in the treatment of burn and scald injuries and determine its bioactive constituents”. Please see lines 2-3 in the manuscript.

What do the authors mean by initial chemical constituents (are they analyzing primary metabolites?) and “figure out the effect” which effect?

Response: Thank you for your comments. The authors used two methods to determine the chemical constituents of Nanocnide lobata. The basic chemical identification methods applied included a variety of color or precipitation reactions using indicator and chromogenic agents. In addition, UPLC‒MS analyses were utilized to determine specific compounds in Nanocnide lobata. The phrase “figure out the effect” was inappropriate and has been deleted from the manuscript. Please see lines 2-3 in the manuscript.

Response: Thank you for your helpful suggestion. Following your advice, the text has been revised to “To identify the most effective fraction of Nanocnide lobata in the treatment of burn and scald injuries and determine its bioactive constituents”. Please see lines 2-3 in the manuscript.

Response: Thank you for your helpful suggestion. The phrases “paper assays” and “test tube assays” were inappropriate and have been deleted from the manuscript. The text has been changed to the following, which is clear and brief: “Chemical identification methods were used to analyze solutions extracted from Nanocnide lobata using petroleum ether, ethyl acetate, n-butanol using a variety of color reactions” Please see lines 4-6 in the manuscript.

In addition, the phrase “petroleum extract-treated group” has been used instead of “petroleum extraction group” throughout the manuscript. Similar revisions to the “ethyl acetate extract-treated group” and “n-butanol extract-treated group” have also been made accordingly.

Which fraction of Nanocnide lobata was subjected to UPLC-MS analysis?

Response: UPLC‒MS analysis was carried out on the total ethanol extract of Nanocnide lobata.

Response: The conclusion has been restructured and the indicated sentence revised. Please see lines 410-419 in the manuscript.

The entire abstract section requires English and scientific language recheck. Many parts are vague and some are mentioned above.

Response: We appreciate your helpful suggestion. According to your suggestion, the entire manuscript has been revised by American Journal Experts (https://www.aje.cn/). Please find the Editing Certificate uploaded as an attachment.

Similarly Introduction section also requires a thorough recheck

Response: The introduction has been revised accordingly.

Line 45: "Long-term fever is prone to various complications and life-threatening" incomplete sentence

Response: The indicated sentence has been revised to the following: “Additionally, long-term fever can easily lead to various complications and further endanger life” .Please see lines 50-51 in the manuscript.

Line 53-54: Please recheck

Response: The indicated sentence has been revised to the following: “Nanocnide lobata is a traditional Chinese medicine (TCM) that is usually used to treat lung heat and cough, scrofula, hemoptysis, burns and scalds, carbuncles, bruises, snakebites, and traumatic bleeding.” Please see lines 59-62 in the manuscript.

Line 56-57. If clinical efficacy has been proven then what are the authors trying to address in the study?

Response: We apologize for any misunderstanding. The indicated text has been revised to the following: “Nanocnide lobata is commonly used to treat burns and scalds in Chinese folk medicine. However, there has been little modern clinical research on the pharmacology and efficacy of Nanocnide lobata in the treatment of burn and scald injuries”. Please see lines 62-65 in the manuscript.

Please recheck Line 65-67.

Response: The indicated sentence has been revised to the following: “Chemical identification methods were utilized to investigate the chemical composition of each extracted fraction of Nanocnide lobata and provide a chemical foundation for further mechanistic research”. Please see lines 71-73 in the manuscript.

Response: We apologize for any misunderstanding. The indicated sentence has been revised to the following: “Then, the petroleum ether extract, ethyl acetate ester extract, and n-butanol extract were each mixed with white Vaseline at a proportion of 85% white Vaseline and 15% extract to obtain ointments of suitable consistency for the further screening of active fractions.” Please see lines 90-93 in the manuscript.

The preparation of ointment may be moved to methods section. Line 134, 137, 140, 143 headings may be restructured. Also provide references for the phytochemical tests performed for every fraction.

Response: Thank you for your suggestion. The description of ointment preparation has been moved to the methods section. In addition, the headings on lines 134, 137, 140, and 143 have been restructured, and references for the phytochemical tests performed for every fraction have been provided.

Line 148: “Preparation of samples” heading may be more appropriate.

Response: The indicated heading has been revised to “Preparation of samples”, as suggested. Please see lines 145 in the manuscript.

Section 3.3 also requires restructuring.

Response: Section 3.3 has been restructured.

Line 231 and 301, reconsider the headings

Response: The headings have been reconsidered and revised.

Discussion: Lines 328, 331-337, 342-348, 360-362, 378-385 require special attention as the scientific and English language are not correct.

Response: Thank you for your advice. These sentences have been revised. Please see the detail of discussion part in the manuscript.

Have the authors identified any bioactive compounds from the UPLC-MS, effective in the treatment of burns and scalds. They may mention them in the discussion section.

Response: Thank you very much for your suggestion. The bioactive compounds identified by UPLC‒MS that are effective in the treatment of burns and scalds have been mentioned in the discussion section. Please see lines 356-371 in the manuscript.

Conclusion section also requires restructuring.

Response: The conclusion section has been restructured accordingly. Please see the detail of conclusion part in the manuscript.

Please recheck the table and figure numbers within text are mentioned appropriately.

Response: Thank you for your helpful suggestion. The table and figure numbers have been rechecked and revised as needed.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(55KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0287147.r005

Decision Letter 2

Sairah Hafeez Kamran

24 May 2023

PONE-D-22-27583R2Investigating the active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald injuriesPLOS ONE

Dear Dr. mao,

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

ACADEMIC EDITOR: Most of the comments of the reviewers have been addressed but few minor changes as suggested below shall be addressed.

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

Please submit your revised manuscript by Jul 08 2023 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:

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We look forward to receiving your revised manuscript.

Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #3: (No Response)

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: Review Report:

The manuscript entitled “Investigating the active chemical constituents and pharmacology of Nanocnide lobate in the treatment of burn and scald injuries” is an interesting study in which authors made attempts to contribute in the field.

Note: I reviewed the file named (PONE-D-22-27583_R2_Pdf) downloaded from “View Submission” and all my comments are according to the line numberings of the pdf file “PONE-D-22-27583_R2_Pdf”

Detailed Comments/Recommendations:

Title:

� Title is well written and clearly indicating the theme of the study.

Abstract:

� It will be more appropriate and impressive if the names of any two to three out of 39 compounds are mentioned in line 23.

� In line 8: Kindly add gander of mice as well.

� It will be more impressive if authors may use few numerical results i.e. (TNF-α and IL-10 or TGF-β1) in line 27 and 28.

Key Words:

� Three out of five key words are already present in the title. It will be more impressive if the authors use the key words other than the words used in Title.

Introduction:

� Overall the introduction is very well written.

� Kindly add family name of Nanocnide lobata anywhere in line 54-56.

Materials and Methods:

� Kindly mention the identification/herbarium number, which was obtained after identification of plant from a botanist for future reference at the end of line 80.

� Line 84: Mention the technique used for evaporation of ethanol, involving standard operating conditions eg. Temperature, Pressure etc.

� Line 116: Kindly mention the light and dark duration and standard feeding of animals as well.

� Line 176: Kindly explain the study design clearly for example how much doses of extracts and reference drugs were administered to animals and by which route and for how many days. All study design should be quite clear for the reader. It will be more appropriate and facilitate the reader to understand if the authors mention the dosing of animals in in tabular format.

� Line 203: Kindly mention how much blood was obtained and from which place eg. Heart etc.

Results & Discussion:

� Results are well expressed.

� Discussion is well written by the authors.

� Line 357: It will be more appropriate if the authors mentions the importance/advantages of UPLC-MS used here in this study.

� Table 6: Kindly mention the details of control, model and positive drug groups. Give descriptions to facilitate the reader. The table should be self-explanatory. Similarly, Tables 7-10 should also be self-explanatory.

� Figures 3-5 are not explained. What does A, B, C, D, E, and F indicate? Histopathological scoring is missing.

� The authors did not discuss the histopathological results.

Conclusion:

� The conclusion is well written.

References:

� The authors are advised to review all the references for the strict adherence of the uniformity of Journal’s references style.

� Reference no 13. Lines 478-480: Reference is incomplete. Kindly add on full details.

� Reference no 34. Lines 544-546: Reference is incomplete. Kindly add on full details.

� Check the font style and font size of references as well. It should be uniform.

Concluding Remarks:

The paper needs above mentioned suggestions along with the English Language proof readings by some language expert. Then after fulfilling the corrections the paper will be acceptable.

**********

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.

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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 #3: Yes: Dr. Aamir Mushtaq

Department of Pharmaceutical Sciences

Government College university Lahore, Pakistan

**********

Attachment

Submitted filename: Review Comments 2nd paper.docx

Click here for additional data file.^{(15KB, docx)}

PLoS One. 2023 Jun 13;18(6):e0287147. doi: 10.1371/journal.pone.0287147.r006

Author response to Decision Letter 2

30 May 2023

Dear editor and reviewer,

Thanks for your letter and comments concerning our manuscript entitled "Investigating active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald injuries" (Manuscript ID PONE-D-22-27583). We really appreciate Plos One for giving us an opportunity to revise our manuscript. The manuscript has been carefully and extensively revised according to your helpful comments. The changes have been highlighted in red in the revised manuscript point by point.

And the responses were as follows:

Detailed Comments/Recommendations:

Title:

�Title is well written and clearly indicating the theme of the study.

Response: Thank you for your help.

Abstract:

�It will be more appropriate and impressive if the names of any two to three out of 39 compounds are mentioned in line 23.

�Response: Following your suggestion, the names of any two to three out of 39 compounds are mentioned in line 23 to line 25. Please check the detail in the manuscript.

�

�In line 8: Kindly add gander of mice as well.

�Response: Do the reviewer means “gender of mice”? The gender of the mice is female which had been provided in the abstract part of the manuscript. Please check in line 8.

�

�It will be more impressive if authors may use few numerical results i.e. (TNF-α and IL-10 or TGF-β1) in line 27 and 28.

�Response: Thanks for your careful work. Few numerical results were listed in line 29 to line 32.

Key Words:

�Three out of five key words are already present in the title. It will be more impressive if the authors use the key words other than the words used in Title.

�Response: The key words and title is partly same. However, if I can’t use the similar word in the key words part. It was inability to summarize or indicate the key points of the article well. Therefore, I suggest the key words may changed to “Nanocnide lobata, chemical composition, UPLC‒MS, preliminary test, burn and scald injuries, pharmacological effects” .Please check in line 41-42.

Introduction:

�Overall the introduction is very well written.]

�Response: Thank for your constructive comments.

�Kindly add family name of Nanocnide lobata anywhere in line 54-56.

�Response: The family name of Nanocnide lobata is Urticaceae which was added in the manuscript. Please check it in line 59 to 62.

Materials and Methods:

�Kindly mention the identification/herbarium number, which was obtained after identification of plant from a botanist for future reference at the end of line 80.

�Response: The original herbarium is kept in the 407 Natural medicinal chemistry Laboratory of the Scientific Research Center of Chongqing Three Gorges Medical College (No.20210503) .Please check it in line 82 to 85.

�

�Line 84: Mention the technique used for evaporation of ethanol, involving standard operating conditions eg. Temperature, Pressure etc.

�Response: Thanks for your careful work. The technique which was used for evaporation of ethanol listed in the manuscript. Please check in line 91.

�

�Line 116: Kindly mention the light and dark duration and standard feeding of animals as well.

�Response: The breeding environment was kept at 25±10℃, the relative humidity was 40% to 70%, and the light-dark cycle was 12 h. Adapt to feeding for consecutive 3 days, fasting 12 h before the experiment, and drinking water freely. Please check in line 123 to 125.

�Line 176: Kindly explain the study design clearly for example how much doses of extracts and reference drugs were administered to animals and by which route and for how many days. All study design should be quite clear for the reader. It will be more appropriate and facilitate the reader to understand if the authors mention the dosing of animals in in tabular format.

�Response: Following your suggestion, the the study design has been revised which is clearly and briefly. Please check in line 192 to line 196. In addition, the table 1 titled “Animal grouping and treatment” was added in the manuscript which mention the dosing of animals in in tabular format.

�

�Line 203: Kindly mention how much blood was obtained and from which place eg. Heart etc.

�Response: Take approximately 1 mL of blood from the orbit of mice, deposit for 20 minutes, then centrifuge at 3000 rpm for 5 minutes, and take the upper serum. Store the serum in a refrigerator at -80 ° C for later use. Please check in line 213 to 216.

Results & Discussion:

�Results are well expressed.

�Response: Thanks for your comments.

�

�Discussion is well written by the authors.

�Response: Thanks for your encouragement.

�

�Line 357: It will be more appropriate if the authors mentions the importance/advantages of UPLC-MS used here in this study.

�Response: The importance/advantages of UPLC-MS used here in this study was presented in the manuscript. Please check it in 376 to 381.

�

�Table 6: Kindly mention the details of control, model and positive drug groups. Give descriptions to facilitate the reader. The table should be self-explanatory. Similarly, Tables 7-10 should also be self-explanatory.

�Response: Thanks for your careful work. The tables 8-11 was revised which mention the details of control, model and positive drug groups.The tables are self-explanatory.

�

�Figures 3-5 are not explained. What does A, B, C, D, E, and F indicate? Histopathological scoring is missing.

�Response: The figures 3-5 has been explained in line 302 to 324, please check. In addition, the histopathological scoring was added in the manuscript. Please check in line 221 to 224 as well as figure 6 (new added).

�

�The authors did not discuss the histopathological results.

�Response: The histopathological results has been discussed in the results part. Please check in line 302 to 329.

�

Conclusion:

�The conclusion is well written.

�Response: Thanks for your comments.

References:

�The authors are advised to review all the references for the strict adherence of the uniformity of Journal’s references style.

�Response: The references style was revised to the style which meet the standards of Plos One.

�Reference no 13. Lines 478-480: Reference is incomplete. Kindly add on full details.

�Response: The full details of references style was added.

�

�Reference no 34. Lines 544-546: Reference is incomplete. Kindly add on full details.

�Response: The full details of references style was added.

�

�Check the font style and font size of references as well. It should be uniform.

�Response: The references style was revised one by one which may meet the standards of Plos One.

Concluding Remarks:

The paper needs above mentioned suggestions along with the English Language proof readings by some language expert. Then after fulfilling the corrections the paper will be acceptable.

Response: Following your suggestion, the entire manuscript was revised by American Journal Experts (https://www.aje.cn/). Please check the English and scientific language certificate which uploaded as the attachment.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(20KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0287147.r007

Decision Letter 3

Sairah Hafeez Kamran

31 May 2023

Investigating the active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald injuries

PONE-D-22-27583R3

Dear Dr. mao,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0287147.r008

Acceptance letter

Sairah Hafeez Kamran

5 Jun 2023

PONE-D-22-27583R3

Investigating the active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald injuries

Dear Dr. Mao:

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.

If we can help with anything else, please email us at plosone@plos.org.

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

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Sairah Hafeez Kamran

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Table. The detailed information of components identified in Nanocnide lobata based on UPLC-MS.

(XLSX)

Click here for additional data file.^{(13.7KB, xlsx)}

S2 Table. The detailed information of contracture rate of wound.

(XLSX)

Click here for additional data file.^{(18.4KB, xlsx)}

S3 Table. The detailed information of ELISA on TNF-α, IL-10, TGF-β1, VEGF respectively.

(XLSX)

Click here for additional data file.^{(12.9KB, xlsx)}

Attachment

Submitted filename: Reviwers Comments.docx

Click here for additional data file.^{(12.7KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(19KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(55KB, docx)}

Attachment

Submitted filename: Review Comments 2nd paper.docx

Click here for additional data file.^{(15KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(20KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Investigating the active chemical constituents and pharmacology of Nanocnide lobata in the treatment of burn and scald injuries

Yanlin Zou

Cao Yu

Qian Huang

Xiaorong Tan

Xiaoyan Tan

Xiaolong Zhu

Dongyang Yi

Jingxin Mao

Roles

Abstract

Objective

Methods

Results

Conclusion

1. Introduction

2. Materials and methods

2.1 Materials

2.2 Reagents

2.3 Experimental animals

2.4 Instruments

2.5 Preliminary chemical identification tests

2.6 Extraction of bioactive ingredients from Nanocnide lobata

2.6.1 Preparation of samples

2.6.2 UPLC‒MS analyses

2.7 Pharmaceutical trial validation

2.7.1 Establishment of the scald/burn model and treatment of mice

Table 1. Animal grouping and treatment.

2.7.2 Measurement of wound healing rate

2.7.3 Histopathological examination

2.7.4 ELISA

2.8 Data analysis

2.9 Ethics statement

3. Results

3.1 Chemical composition categories for each extraction fraction

Table 2. Identification test of chemical composition of petroleum ether extraction part on Nanocnide lobata.

Table 3. Identification test of chemical composition of ethyl acetate extraction part on Nanocnide lobata.

Table 4. Identification test of chemical composition of n-butanol extraction part on Nanocnide lobata.

Table 5. Identification test of chemical composition of water extraction part on Nanocnide lobata.

3.2 UPLC‒MS results

Fig 1.

Table 6. The typical total ion chromatogram of Nanocnide lobata sample in positive ion mode and negative ion mode respectively.

3.3 Observation of scald/burn wounds

Fig 2. General observation of the wound.

3.4 Wound healing rate results

Table 7. Change in percent contracture rate of wound (x¯±s).

3.5 HE staining results

Fig 3. Pathologic sections of wound of mice in day 7.

Fig 4. Pathologic sections of wound of mice in day 14.

Fig 5. Pathologic sections of wound of mice in day 21.

Fig 6. The histopathological scores of the burn and scald injuries in the mice after treated with Nanocnide lobate.

3.5 ELISA results

Table 8. Changes in the concentration of TNF-α in each group at different time points after injury (x¯±s).

Table 9. Changes in the concentration of IL-10 in each group at different time points after injury (x¯±s).

Table 10. Changes in the concentration of TGF-β1 in each group at different time points after injury (x¯±s).

Table 11. Changes in the concentration of VEGF in each group at different time points after injury (x¯±s).

4. Discussion

5. Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Sairah Hafeez Kamran

Roles

Author response to Decision Letter 0

Decision Letter 1

Sairah Hafeez Kamran

Roles

Author response to Decision Letter 1

Decision Letter 2

Sairah Hafeez Kamran

Roles

Author response to Decision Letter 2

Decision Letter 3

Sairah Hafeez Kamran

Roles

Acceptance letter

Table 7. Change in percent contracture rate of wound ( $\bar{x} \pm s$ ).

Table 8. Changes in the concentration of TNF-α in each group at different time points after injury ( $\bar{x} \pm s$ ).

Table 9. Changes in the concentration of IL-10 in each group at different time points after injury ( $\bar{x} \pm s$ ).

Table 10. Changes in the concentration of TGF-β1 in each group at different time points after injury ( $\bar{x} \pm s$ ).

Table 11. Changes in the concentration of VEGF in each group at different time points after injury ( $\bar{x} \pm s$ ).