2种化学型的黄花蒿萜类代谢物及其相关基因表达分析

王婷婷; 戴仕林; 刘潺潺; 蒋征; 吴啟南

doi:10.14148/j.issn.1672-0482.2022.0770

2种化学型的黄花蒿萜类代谢物及其相关基因表达分析

doi: 10.14148/j.issn.1672-0482.2022.0770

王婷婷^{1, 2,},
戴仕林^{1, 2},
刘潺潺^{1, 2},
蒋征¹,
吴啟南^{1, 2, 3, ,}

1.
南京中医药大学药学院, 江苏南京 210023
2.
江苏省中药资源产业化过程协同创新中心, 江苏南京 210023
3.
中药资源产业化与方剂创新药物国家地方联合工程研究中心, 江苏南京 210023

基金项目:

国家中医药管理局专项国家中药标准化项目 ZYBZH-C-JS-31

国家中医药管理局道地药材生态种植及质量保障项目 2021

详细信息

作者简介:
王婷婷, 女, 硕士研究生, E-mail: wangtingt816@163.com

吴啟南，1964年生，男，江苏南通人。南京中医药大学教授，博士生导师，江苏省中药资源产业化过程协同创新中心副主任。江苏省高等学校中药资源化学科技创新团队带头人，国家一流专业中药资源与开发专业、国家级一流课程中药鉴定虚拟仿真实验项目负责人。兼任教育部高等学校中药学类专业教指委委员、中国自然资源学会中药及天然药物资源专业委员会副主任兼秘书长、世中联中药鉴定专业委员会副主任委员、中华中医药学会中药鉴定分会副主任委员等。曾获全国优秀科技工作者、江苏省“五一”劳动奖章等。长期从事中药资源生产、中药鉴定及药材品质形成机制等相关领域的教学与科研工作。主持国家科技支撑计划、国家自然科学基金等10余项国家级课题; 在国内外学术期刊上发表论文200余篇，其中SCI论文40余篇; 授权专利11项; 获国家科技进步奖二等奖1项、省级科技进步奖3项。主编教材或专著5部，主编的专著《新编中国药材学》获第五届中国出版政府奖; 获省级教学成果二等奖2项

通讯作者:
吴啟南, 男, 教授, 博士生导师, 主要从事道地药材生产与品质评价研究, E-mail: wuqn@njucm.edu.cn

中图分类号: R284
计量
- 文章访问数: 187
- HTML全文浏览量: 33
- PDF下载量: 8
- 被引次数: 0
出版历程
- 收稿日期: 2022-01-22
- 网络出版日期: 2022-09-19
- 发布日期: 2022-09-10

Expression Analysis of Two Chemical-Types of Artemisia Annua L. Terpenoids and Their Related Genes

WANG Ting-ting^{1, 2
,},
DAI Shi-lin^{1, 2},
LIU Chan-chan^{1, 2},
JIANG Zheng¹,
WU Qi-nan^{1, 2, 3
, ,}

1.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China
3.
National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing 210023, China

摘要

摘要: 目的分析2种化学型黄花蒿萜类代谢物及其转录组数据信息, 探讨2种化学型黄花蒿差异代谢物及其可能形成原因。方法采集江苏盱眙县、河南中牟县两地黄花蒿种子于同一条件下种植, 通过顶空-气相色谱-三重四极杆串联质谱(HS-GC-QQQ-MS/MS)分析黄花蒿的挥发性成分, 超快速高效液相色谱-三重四级杆飞行时间串联质谱(UFLC-Triple TOF-MS/MS)非靶向分析2种化学型黄花蒿的代谢物, 并采用转录组测序分析挥发性成分相关萜类生物合成基因的表达。结果依据挥发性主成分类别分型, 江苏盱眙县种源黄花蒿为蒿酮型, 河南中牟县种源黄花蒿为樟脑型。UFLC-Triple TOF-MS/MS检测到的差异代谢物通过KEGG数据库分析可知蒿酮型和樟脑型黄花蒿分别在倍半萜、三萜生物合成和二萜生物合成路径显著上调。转录组数据中通向萜类骨架生物合成的甲基赤藓糖醇-4-磷酸(MEP)途径和甲羟戊酸(MVA)途径注释到11个关键酶的23个候选基因具有显著差异。单萜合成路径中, 1, 8-桉树脑合酶(TPS-Cin)共检测到10个候选基因。此外, 还发现2个冰片脱氢酶候选基因在樟脑型黄花蒿中高表达, 3个黄花蒿醇脱氢酶2(ADH2)候选基因在蒿酮型黄花蒿中高表达。结论该研究为揭示黄花蒿化学型形成分子机制提供科学数据。
- 黄花蒿 /
- 挥发性成分 /
- 化学型 /
- 萜类代谢物 /
- 生物合成
Abstract: OBJECTIVE To investigate the differential metabolites of two chemical-types of Artemisia annua and their possible causes by analyzing the data of terpenoid metabolites and their transcriptome of two chemical-types of Artemisia annua. METHODS The seeds of Artemisia annua were collected from Xuyi County, Jiangsu Province, and Zhongmou County, Henan Province, in China, and grown under the same conditions. The volatile components of Artemisia annua were analyzed by headspace-gas chromatography-triple quadrupole mass spectrometry (HS-GC-QQQ-MS/MS), non-targeted analysis of the differential metabolites of 2 chemical-types of Artemisia annua were performed by ultra-fast liquid chromatography-triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS), and the expression of the biosynthetic genes of the volatile components was analyzed by transcriptome sequencing. RESULTS Based on the typing of volatile components, Artemisia annua from Xuyi County, Jiangsu Province was classified as artemisia ketone type, and Artemisia annua from Zhongmou County, Henan Province was classified as camphor type. The differential metabolites detected by UFLC-Triple TOF-MS/MS were significantly up-regulated in sesquiterpene, triterpene, and diterpene biosynthesis pathways by Kyoto encyclopedia of genes and genomes (KEGG) database analysis for artemisia ketone type and camphor types of Artemisia annua, respectively. The 2-methyl-D-erythritol-4-phosphate (MEP) pathway leading to terpene skeleton biosynthesis and the mevalonate (MVA) pathway annotated 23 candidate genes for 11 key enzymes that were significantly differential in the transcriptome data. In the monoterpene synthesis pathway, 10 candidate genes were detected for 1, 8-cineole synthase (TPS-Cin). In addition, two borneol dehydrogenase candidates were found to be highly expressed in camphor-type Artemisia annua, and three Artemisia annua alcohol dehydrogenase (ADH2) candidates were highly expressed in artemisia ketone-type Artemisia annua. CONCLUSION This study provides scientific data to reveal the molecular mechanism of Artemisia annua chemical-types formation.
- Artemisia annua L. /
- volatile components /
- chemical type /
- terpenoids /
- biosynthesis

HTML全文

图 1 黄花蒿HS-GC-QQQ-MS/MS总离子流色谱图

注: A.JXAa; B.HZAa

Figure 1. Total ion chromatogram of Artemisia annua HS-GC-QQQ-MS/MS

下载: 全尺寸图片幻灯片

图 2 蒿酮型黄花蒿UFLC-Triple TOF-MS/MS基峰图

注: A.正离子模式; B.负离子模式

Figure 2. UFLC-Triple TOF-MS/MS base peak of ketone type Artemisia annua

下载: 全尺寸图片幻灯片

图 3 樟脑型黄花蒿UFLC-Triple TOF-MS/MS基峰图

注: A.正离子模式; B.负离子模式

Figure 3. UFLC-Triple TOF-MS/MS base peak of camphor type Artemisia annua

下载: 全尺寸图片幻灯片

图 4 正离子模式下2种化学型黄花蒿PCA图(A)、OPLS-DA得分图(B)、OPLS-DA模型验证图(C)

Figure 4. PCA plots (A), OPLS-DA score plots (B) and OPLS-DA model validation plots (C) of two chemical types of Artemisia annua in positive ion mode

下载: 全尺寸图片幻灯片

图 5 负离子模式下2种化学型黄花蒿PCA图(A)、OPLS-DA得分图(B)、OPLS-DA模型验证图(C)

Figure 5. PCA plots (A), OPLS-DA score plots (B) and OPLS-DA model validation plots (C) of two chemical types of Artemisia annua in negative ion mode

下载: 全尺寸图片幻灯片

图 6 蒿酮型(A)和樟脑型(B)黄花蒿上调代谢物通路分析

Figure 6. Analysis of the up-regulated metabolite pathway of artemisia ketone type (A) and camphor type (B) Artemisia annua

下载: 全尺寸图片幻灯片

图 7 2种化学型黄花蒿差异基因火山图(A)和差异基因GO分类图(B)

Figure 7. Differential gene volcano map (A) and gene GO classification map (B) of two chemical types of Artemisia annua

下载: 全尺寸图片幻灯片

图 8 2种化学型黄花蒿挥发性成分相关萜类合成通路基因表达

注: AK-T.蒿酮型; C-T.樟脑型; DXS.1-脱氧-D-木酮糖-5-磷酸合成酶; HDR.4-羟基-2-甲基-2-E-丁烯基-4-焦磷酸还原酶; IPPI.异戊烯基焦磷酸异构酶; GPS.异戊烯基转移酶牻牛儿基焦磷酸合酶; GGPS.牻牛儿基牻牛儿基焦磷酸合成酶; AACT.乙酰辅酶A酰基转移酶; HMGR.3-羟基-3-甲基戊二酰辅酶A还原酶; MK.甲羟戊酸激酶; PMK.甲羟戊酸激酶; MPDC.甲羟戊酸焦磷酸脱羧酶; FDPS.法尼基焦磷酸合酶; CPS.内根-古巴焦磷酸合成酶; GA3.贝壳杉烯C19氧化酶; GA20OX.赤霉素双加氧酶; GA3OX.赤霉素3-β-双氧酶; TPS04.香叶基芳樟醇合酶; CYP82G1.三甲基十三烷四烯合酶; ADH2.黄花蒿醇脱氢酶2;TPS-Cin.1, 8-桉树脑合酶

Figure 8. Gene expression of terpenoid synthesis pathway related to volatile components of 2 chemical types of Artemisia annua

下载: 全尺寸图片幻灯片

表 1 黄花蒿挥发性成分(n=4)

Table 1. List of volatile components of Artemisia annua samples(n=4)

编号	CAS	英文名称	中文名称	峰面积百分比/%
编号	CAS	英文名称	中文名称	JXAa	HZAa
1	3208-16-0	2-Ethylfuran	2-乙基呋喃	1.87±1.2	0.7±0.32
2	66-25-1	Hexanal	正己醛	2.95±1.63	0.92±0.23
3	505-57-7	2-Hexenal	2-己烯醛	3.35±2.15	1.51±0.57
4	544-12-7	trans-3-Hexen-1-ol	反式-3-己烯-1-醇	0.91±0.46	0.55±0.12
5	13466-78-9	3-Carene	3-蒈烯	1.9±1.25	4.55±0.59
6	79-92-5	Camphene	莰烯	5.46±5.25	21.38±0.96
7	555-10-2	β-Phellandrene	3-异丙基-6-亚甲基-1-环己烯	-	1.13±0.4
8	99-84-3	β-Terpinene	对薄荷-1(7), 3-二烯	6.28±1.08	5.65±2.48
9	99-86-5	α-Terpinene	松油烯	-	0.37±0.05
10	527-84-4	o-Cymene	邻伞花烃	0.46±0.09	1.28±0.13
11	470-82-6	Eucalyptol	桉叶油醇	12.37±3.45	21.17±1.51
12	99-85-4	γ-Terpinene	γ-松油烯	-	0.73±0.15
13	546-49-6	Artemisia ketone	蒿酮	51.28±8.31	-
14	17699-16-0	4-Thujanol	4-侧柏醇	-	1.13±0.57
15	15537-55-0	cis-(+/-)-4-Thujanol	顺-(+/-)-4-侧柏醇	-	0.43±0.12
16	76-22-2	Camphor	樟脑	6.8±6.14	30.7±4.44
17	30460-92-5	Pinocarvone	松香芹酮	-	0.61±0.09
18	564-94-3	Myrtenal	桃金娘烯醛	-	0.45±0.14
19	56423-40-6	Butanoic acid, 2-methyl-, phenylmethyl ester	2-甲基丁酸苯甲酯	0.52±0.23	-
20	87-44-5	β-Caryophyllene	β-石竹烯	0.99±0.47	0.4±0.16
21	92692-39-2	Amorpha-4, 11-diene	青蒿二烯	0.53±0.17	-
22	54274-73-6	epi-Bicyclosesquiphellandrene	epi-双环倍半水芹烯	1.57±1.21	0.7±0.41
注: -表示未检测出该成分。

下载: 导出CSV

表 2 樟脑型黄花蒿二萜通路上调差异代谢物

Table 2. Up-regulated differential metabolites in camphor type Artemisia annua diterpene pathway

序号	保留时间/min	分子式	质荷比m/z	化合物名称	加和离子	差异倍数	P值	VIP
1	8.565	C₂₀H₃₀O₃	275.237	ent-7α-Hydroxykaur-16-en-19-oic acid	[M-CO₂+H]⁺	0.056	3.896E-07	1.464
2	6.768	C₂₀H₃₂O	307.264	Kaur-16-en-18-ol	[M+H₂O+H]⁺	0.072	9.921E-05	1.453
3	7.539	C₂₀H₃₀O	305.247	Kaur-16-en-18-al	[M+H₂O+H]⁺	0.081	2.691E-03	1.401
4	7.953	C₂₀H₃₄O	291.269	香叶基芳樟醇[(E, E)-Geranyllinalool]	[M+H]⁺	0.289	5.714E-03	1.372

下载: 导出CSV

参考文献(34)

[1]	ROVESTI P. Essential oils of some chemotypes of aromatic Eritrean labiates[J]. Pharm Weekbl, 1957, 92(23): 843-845.
[2]	智亚楠, 陈月华, 马宇明, 等. 河南省黄花蒿挥发油的组分分析及抑菌作用测定[J]. 河南农业科学, 2016, 45(12): 105-109. https://www.cnki.com.cn/Article/CJFDTOTAL-HNNY201612022.htm ZHI YN, CHEN YH, MA YM, et al. The chemical constituents and antifungal activity of essential oil from Artemisia annua linn in Henan Province[J]. J Henan Agric Sci, 2016, 45(12): 105-109. https://www.cnki.com.cn/Article/CJFDTOTAL-HNNY201612022.htm
[3]	SANTOMAURO F, DONATO R, PINI G, et al. Liquid and vapor-phase activity of Artemisia annua essential oil against pathogenic Malassezia spp[J]. Planta Med, 2018, 84(3): 160-167. doi: 10.1055/s-0043-118912
[4]	BILIA AR, SANTOMAURO F, SACCO C, et al. Essential oil of Artemisia annua L. : An extraordinary component with numerous antimicrobial properties[J]. Evid Based Complement Alternat Med, 2014, 2014: 159819.
[5]	赖镇源. 青蒿挥发油抗痤疮病原体及解热作用的实验研究[D]. 广州: 广州中医药大学, 2005. LAI ZY. The experimental study of anti-acne pathogeny and defervescence of Artemisia naphtha[D]. Guangzhou: Guangzhou University of Chinese Medicine, 2005.
[6]	RASHID S, RATHER MA, SHAH WA, et al. Chemical composition, antimicrobial, cytotoxic and antioxidant activities of the essential oil of Artemisia indica Willd[J]. Food Chem, 2013, 138(1): 693-700. doi: 10.1016/j.foodchem.2012.10.102
[7]	SOKTOEVA TE, RYZHOVA GL, DYCHKO KA, et al. Artemisinin content in Artemisia annua L. extracts obtained by different methods[J]. Russ J Bioorg Chem, 2013, 39(7): 761-764. doi: 10.1134/S1068162012070199
[8]	ZHIGZHITZHAPOVA SV, DYLENOVA EP, GULYAEV SM, et al. Composition and antioxidant activity of the essential oil of Artemisia annua L[J]. Nat Prod Res, 2020, 34(18): 2668-2671. doi: 10.1080/14786419.2018.1548461
[9]	FENG XC, CAO SJ, QIU F, et al. Traditional application and modern pharmacological research of Artemisia annua L[J]. Pharmacol Ther, 2020, 216: 107650. doi: 10.1016/j.pharmthera.2020.107650
[10]	张小波, 赵宇平, 黄晓巍, 等. 青蒿道地药材研究综述[J]. 中国中药杂志, 2016, 41(11): 2015-2018. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZY201611007.htm ZHANG XB, ZHAO YP, HUANG XW, et al. Review on study of Dao-di herbs Artemisiae Annuae Herba[J]. China J Chin Mater Med, 2016, 41(11): 2015-2018. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZY201611007.htm
[11]	朱亚莹. 热毒宁注射液原料药青蒿的品质评价研究[D]. 南京: 南京中医药大学, 2020. ZHU YY. Study on quality evaluation of artemisiae annuae herba, the raw material of reduning injection[D]. Nanjing: Nanjing University of Chinese Medicine, 2020.
[12]	TSUGAWA H, CAJKA T, KIND T, et al. MS-DIAL: Data-independent MS/MS deconvolution for comprehensive metabolome analysis[J]. Nat Methods, 2015, 12(6): 523-526. doi: 10.1038/nmeth.3393
[13]	PANG ZQ, CHONG J, ZHOU GY, et al. MetaboAnalyst 5.0: Narrowing the gap between raw spectra and functional insights[J]. Nucleic Acids Res, 2021, 49(W1): W388-W396. doi: 10.1093/nar/gkab382
[14]	CHEN CJ, CHEN H, ZHANG Y, et al. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Mol Plant, 2020, 13(8): 1194-1202. doi: 10.1016/j.molp.2020.06.009
[15]	SHEN Q, ZHANG LD, LIAO ZH, et al. The genome of Artemisia annua provides insight into the evolution of Asteraceae family and artemisinin biosynthesis[J]. Mol Plant, 2018, 11(6): 776-788. doi: 10.1016/j.molp.2018.03.015
[16]	LOVE MI, HUBER W, ANDERS S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biol, 2014, 15(12): 550. doi: 10.1186/s13059-014-0550-8
[17]	UMLAUF D, ZAPP J, BECKER H, et al. Biosynthesis of the irregular monoterpene Artemisia ketone, the sesquiterpene germacrene D and other isoprenoids in Tanacetum vulgare L. (Asteraceae)[J]. Phytochemistry, 2004, 65(17): 2463-2470. doi: 10.1016/j.phytochem.2004.08.019
[18]	李洪梅, 黄璐琦, 周爱香, 等. 不同化学型樟对大鼠佐剂性关节炎模型的影响[J]. 中国中药杂志, 2009, 34(24): 3251-3254. doi: 10.3321/j.issn:1001-5302.2009.24.026 LI HM, HUANG LQ, ZHOU AX, et al. Study on antiinflammatory effect of different chemotype of Cinnamomum camphora on rat arthritis model induced by Freund's adjuvant[J]. China J Chin Mater Med, 2009, 34(24): 3251-3254. doi: 10.3321/j.issn:1001-5302.2009.24.026
[19]	MONTERO-VILLEGAS S, CRESPO R, RODENAK-KLADNIEW B, et al. Cytotoxic effects of essential oils from four Lippia alba chemotypes in human liver and lung cancer cell lines[J]. J Essent Oil Res, 2018, 30(3): 167-181. doi: 10.1080/10412905.2018.1431966
[20]	ACIMOVICM, JEREMICJS, TODOSIJEVIC M, et al. Comparative study of the essential oil and hydrosol composition of sweet wormwood (Artemisia annua L. ) from Serbia[J]. Chem Biodivers, 2022, 19(3): e202100954.
[21]	JUTEAU F, MASOTTI V, BESSIERE JM, et al. Antibacterial and antioxidant activities of Artemisia annua essential oil[J]. Fitoterapia, 2002, 73(6): 532-535. doi: 10.1016/S0367-326X(02)00175-2
[22]	CAVAR S, MAKSIMOVIC M, VIDIC D, et al. Chemical composition and antioxidant and antimicrobial activity of essential oil of Artemisia annua L. from Bosnia[J]. Ind Crops Prod, 2012, 37(1): 479-485. doi: 10.1016/j.indcrop.2011.07.024
[23]	SUGA T, SHISHIBORI T, KOTERA K, et al. The biosynthesis of a non-head to tail monoterpene, Artemisia ketone[J]. Chem Lett, 1972, 1(7): 533-534. doi: 10.1246/cl.1972.533
[24]	ALLEN KG, BANTHORPE DV, CHARLWOOD BV, et al. Biosynthesis of Artemisia ketone in higher plants[J]. Phytochemistry, 1977, 16(1): 79-83. doi: 10.1016/0031-9422(77)83018-5
[25]	吕宗友. 抑制青蒿素合成竞争支路及水杨酸调控青蒿中青蒿素合成的研究[D]. 上海: 上海交通大学, 2016. LYU ZY. The study of regulating artemisinin biosynthesis in Artemisia annua by branch pathway blocking and salicylic acid[D]. Shanghai: Shanghai Jiao Tong University, 2016.
[26]	POLICHUK DR, ZHANG YS, REED DW, et al. A glandular trichome-specific monoterpene alcohol dehydrogenase from Artemisia annua[J]. Phytochemistry, 2010, 71(11/12): 1264-1269.
[27]	YAMAGUCHI S. Gibberellin metabolism and its regulation[J]. Annu Rev Plant Biol, 2008, 59: 225-251. doi: 10.1146/annurev.arplant.59.032607.092804
[28]	DAVIERE JM, ACHARD P. Gibberellin signaling in plants[J]. Development, 2013, 140(6): 1147-1151. doi: 10.1242/dev.087650
[29]	COLEBROOK EH, THOMAS SG, PHILLIPS AL, et al. The role of gibberellin signalling in plant responses to abiotic stress[J]. J Exp Biol, 2014, 217(Pt 1): 67-75.
[30]	DU Q, LI CL, LI DQ, et al. Genome-wide analysis, molecular cloning and expression profiling reveal tissue-specifically expressed, feedback-regulated, stress-responsive and alternatively spliced novel genes involved in gibberellin metabolism in Salvia miltiorrhiza[J]. BMC Genomics, 2015, 16: 1087. doi: 10.1186/s12864-015-2315-5
[31]	MA TY, GAO H, ZHANG D, et al. Transcriptome analyses revealed the ultraviolet B irradiation and phytohormone gibberellins coordinately promoted the accumulation of artemisinin in Artemisia annua L[J]. Chin Med, 2020, 15: 67. doi: 10.1186/s13020-020-00344-8
[32]	张铁军, 王于方, 刘丹, 等. 天然药物化学史话: 青蒿素——中药研究的丰碑[J]. 中草药, 2016, 47(19): 3351-3361. doi: 10.7501/j.issn.0253-2670.2016.19.001 ZHANG TJ, WANG YF, LIU D, et al. Historical story on natural medicinal chemistry: Artemisinin, a milestone of traditional Chinese medicine study[J]. Chin Tradit Herb Drugs, 2016, 47(19): 3351-3361. doi: 10.7501/j.issn.0253-2670.2016.19.001
[33]	彭琼辉, 刘琼, 刘成高, 等. 热毒宁注射液治疗AECOPD的疗效及对TLR4/NF-κB炎症信号通路的影响[J]. 湖南中医杂志, 2021, 37(12): 1-4, 20. https://www.cnki.com.cn/Article/CJFDTOTAL-HNZO202112001.htm PENG QH, LIU Q, LIU CG, et al. Clinical effect of Reduning injection in treatment of acute exacerbation of chronic obstructive pulmonary disease and its effect on the Toll-like receptor 4/nuclear factor-kappa B inflammatory signaling pathway[J]. Hunan J Tradit Chin Med, 2021, 37(12): 1-4, 20. https://www.cnki.com.cn/Article/CJFDTOTAL-HNZO202112001.htm
[34]	罗杰平, 黄胜贤, 陈丽珍, 等. 甘草酸苷联合热毒宁治疗手足口病患儿的临床疗效及对免疫反应的影响[J]. 川北医学院学报, 2022, 37(1): 35-38. https://www.cnki.com.cn/Article/CJFDTOTAL-NOTH202201008.htm LUO JP, HUANG SX, CHEN LZ, et al. Clinical efficacy of Reduning combined with glycyrrhizin in the treatment of children with hand-foot-mouth disease and its effect on inflammatory immune reaction[J]. J North Sichuan Med Coll, 2022, 37(1): 35-38. https://www.cnki.com.cn/Article/CJFDTOTAL-NOTH202201008.htm