水牛角含巯基类成分解热效应评价研究

黄思莹; 冯旗园; 包旺林; 黄潇正; 武文星; 赵明; 段金廒; 刘睿

doi:10.14148/j.issn.1672-0482.2024.0268

水牛角含巯基类成分解热效应评价研究

doi: 10.14148/j.issn.1672-0482.2024.0268

黄思莹^1,,
冯旗园¹,
包旺林¹,
黄潇正¹,
武文星¹,
赵明^{1, 2},
段金廒^{1, 2, ,},
刘睿^{1, 2, ,}

1.
南京中医药大学/江苏省中药资源产业化过程协同创新中心/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/国家中医药管理局中药资源循环利用重点研究室, 江苏南京 210023
2.
动物类中药与功能肽国际合作联合实验室, 江苏南京 210023

基金项目:

国家自然科学基金面上项目 81973450

江苏省高校“青蓝工程”中青年学术带头人项目

江苏省农业科技自主创新项目 CX(22)3173

南京中医药大学中药学一流学科开放课题 2020YLXK009

详细信息

作者简介:
黄思莹, 女, 硕士研究生, E-mail: hsyww1231@163.com

通讯作者:
段金廒, 男, 教授, 博士生导师, 主要从事中药资源化学与方剂功效物质基础研究, E-mail: dja@njucm.edu.cn

刘睿, 男, 教授, 博士生导师, 主要从事动物药资源化学研究, E-mail: liurui@njucm.edu.cn

中图分类号: R285.5
计量
- 文章访问数: 59
- HTML全文浏览量: 9
- PDF下载量: 7
- 被引次数: 0
出版历程
- 收稿日期: 2023-12-22
- 网络出版日期: 2024-03-15
- 发布日期: 2024-03-10

Antipyretic Activity of Sulfhydryl Active Fractions Extracted From Bubali Cornu

HUANG Siying^1
,,
FENG Qiyuan¹,
BAO Wanglin¹,
HUANG Xiaozheng¹,
WU Wenxing¹,
ZHAO Ming^{1, 2},
DUAN Jinao^{1, 2
, ,},
LIU Rui^{1, 2
, ,}

1.
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Key Laboratory of Chinese Medicinal Resources Recycling Utilization, State Administration of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Animal-Derived Chinese Medicine and Functional Peptides International Collaboration Joint Laboratory, Nanjing 210023, China

摘要

摘要: 目的制备水牛角含巯基类成分(-SH active fractions, SHF), 评价其解热效应。方法通过十二烷基硫酸钠-二硫苏糖醇(SDS-DTT)联合提取法制备水牛角SHF, 并采用Ellman法测定游离巯基(-SH)浓度; 基于nano LC-MS/MS分析表征水牛角SHF物质组成。采用脂多糖(LPS)建立大鼠发热模型评价水牛角SHF解热活性, 采用酶联免疫吸附法(ELISA)试剂盒测定血浆中前列腺素E₂(PGE₂)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的水平, 测定下丘脑环磷酸腺苷(cAMP)、PGE₂、TNF-α的水平; 采用非靶向代谢组学的方法, 探究水牛角SHF对发热大鼠血浆代谢物干预情况。结果 SDS-DTT联合提取法可有效制备水牛角SHF, 其中主要成分为含丰富半胱氨酸残基(Cysteine, Cys)的Ⅰ型、Ⅱ型角蛋白、角蛋白相关蛋白等, SHF的-SH占蛋白质比例较水牛角水提液提高了约20倍。发热大鼠给予水牛角SHF 0.5 h后大鼠体温显著下降(P < 0.01), 且解热效应持续4.5 h; 同时显著降低血浆及下丘脑PGE₂、IL-1β、TNF-α、cAMP等水平。从空白组与模型组血浆样本中共鉴定出137个潜在差异代谢物, 水牛角SHF可回调其中31个代谢物, 包括溶血磷脂酸、磷脂酰肌醇、磷脂酸、甘油三酯、磷脂酰胆碱等, 主要涉及甘油磷脂代谢通路。结论水牛角SHF解热效应确切, 给药剂量为水提液的1/10即可显示与其相当的解热效应, 为水牛角清热功效物质基础研究提供了方向和依据。
- 水牛角 /
- 含巯基类 /
- 解热 /
- 抗炎 /
- 非靶向代谢组
Abstract: OBJECTIVE To extract the -SH active fractions (SHF) from Bubali Cornu (water buffalo horn) and evaluate its antipyretic activity. METHODS SHF was extracted from Bubali Cornu by SDS-DTT, and the content of native thiols (-SH) was determined by Ellman reagent method. SHF was identified based on nano LC-MS/MS technology. Evaluation of antipyretic activity of SHF was based on LPS-induced fever rat model. The levels of PGE₂, IL-1β, and TNF-α in plasma as well as the levels of cAMP, PGE₂, and TNF-α in the hypothalamus were measured by ELISA kits. An untargeted metabolomics approach was used to further investigate the intervention of SHF on plasma metabolites in febrile rats. RESULTS SDS-DTT could effectively extract SHF from Bubali Cornu, in which the main components were type Ⅰ, type Ⅱ keratins and keratin-associated proteins, which were rich in Cys, and the ratio of -SH to protein in SHF was increased about 20 times more than that of traditional decoction. SHF could significantly decrease (P < 0.01) the body temperature which lasted for 4.5 hours. SHF could also significantly decrease the levels of PGE₂, IL-1β, TNF-α and cAMP in plasma and hypothalamic. A total of 137 potentially differential metabolites were identified from plasma samples of the control and model groups, of which 31 metabolites could be dialed back after SHF administration, including lysophosphatidic acid, phosphatidylinositol, phosphatidic acid, triglycerides, phosphatidylcholine and so on, which were mainly involved in the glycerophospholipid metabolic pathway. CONCLUSION SHF has precise antipyretic effect, and the dosage of 1/10 of the aqueous extract can show its comparable antipyretic effect, which provides the direction and basis for the basic research on the antipyretic efficacy of Bubali Cornu.
- Bubali Cornu /
- -SH active fractions /
- antipyretic /
- anti-inflammatory /
- non-targeted metabolome

HTML全文

图 1 水牛角SHF和水提液的蛋白质种类

Figure 1. Protein types in extract of Bubali Cornu

下载: 全尺寸图片幻灯片

图 2 SHF对发热大鼠血浆中PGE₂、IL-1β、TNF-α及下丘脑组织中cAMP、PGE₂、TNF-α水平的影响

注：与空白组相比, ^###P < 0.001;与模型组相比, ^{* *}P < 0.01, ^{* * *}P < 0.001。x±s, n=8。

Figure 2. Effect of SHF on the levels of PGE₂, IL-1β, TNF-α in plasma and cAMP, PGE₂, TNF-α in hypothalamic tissues of febrile rats

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图 3 正(A)、负(B)离子模式下UPLC-Q-TOF-MS数据的多元统计分析

注：C.空白组; M.模型组; S.水牛角SHF组; A1, B1.PCA分析得分图; A2, B2.OPLS-DA图; A3, B3.OPLS-DA模型置换检验图

Figure 3. Multivariate statistical analysis of UPLC-Q-TOF-MS data in positive (A) and negative (B) ion modes

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图 4 发热相关代谢通路(A)和水牛角SHF解热相关代谢通路(B)

Figure 4. Febrile metabolic pathway (A) and antipyretic metabolic pathway of SHF (B)

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表 1 水牛角水提液和SHF中蛋白和游离-SH含量(x±s, n=3)

Table 1. Protein and free -SH levels in extract of Bubali Cornu(x±s, n=3)

样品	游离巯基浓度/(mmol·L^-1)	蛋白浓度/(g·L^-1)	游离-SH浓度/蛋白浓度/(mmol·g^-1)
水牛角水提液	1.618±0.011	64.505±2.749	0.025±0.001
水牛角SHF	42.188±0.530	86.029±2.417	0.491±0.019

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表 2 水牛角SHF的蛋白信息

Table 2. Protein information for SHF in Bubali Cornu

序号	蛋白名称	覆盖率/%	唯一肽段数量	总肽段数量	含-SH肽段数量
1	Keratin-associated protein 3-3	39	2	5	3
2	Keratin-associated protein 3-1	36	2	12	1
3	Keratin, type Ⅰ microfibrillar 48 kDa, component 8C-1	25	8	28	20
4	Keratin, type Ⅱ cuticular Hb1	23	5	18	5
5	Keratin, type Ⅱ cuticular Hb3	20	10	22	15
6	Keratin, high-sulfur matrix protein, ⅢA3	20	4	4	4
7	Keratin, type Ⅰ microfibrillar, 47.6 kDa	19	3	22	13
8	Keratin, type Ⅱ microfibrillar, component 5	18	6	13	4
9	Collagen alpha-1(Ⅰ) chain	17	22	26	-
10	Keratin, type Ⅰ cuticular Ha5	15	4	20	12
11	Ubiquitin-ribosomal protein eL40 fusion protein	15	4	4	-
12	Chymotrypsin-like elastase family member 2A	14	6	6	-
13	Keratin, type Ⅱ cytoskeletal 5	9	19	15	12
14	Collagen alpha-1(Ⅲ) chain	9	15	19	-
15	Collagen alpha-2(Ⅰ) chain	8	9	9	-
16	Heat shock protein beta-1	8	2	2	-
17	Desmocollin-1	4	3	2	1
18	Peroxiredoxin-6	4	2	4	-
19	Desmoglein-1	3	4	4	-

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表 3 各组大鼠体温变化(x±s, ℃, n=10)

Table 3. Changes in body temperature of rats in each group(x±s, ℃, n=10)

分组	剂量/(g·kg^-1)	基础体温	造模后5.5 h	给药0.5 h	给药1 h	给药1.5 h
空白组		37.36±0.20	36.97±0.20	36.80±0.15	36.82±0.25	36.84±0.15
模型组		37.73±0.30	39.17±0.23^###	39.17±0.37^###	39.16±0.37^###	39.08±0.17^###
阿司匹林组	0.10	37.73±0.27	39.05±0.23^###	38.53±0.16^**	37.78±0.30^***	37.88±0.31^***
水牛角SHF低剂量组	0.312 5	37.72±0.27	39.05±0.31^###	38.78±0.19	38.43±0.44^***	38.37±0.51^***
水牛角SHF中剂量组	0.625	37.91±0.13	39.11±0.17^###	38.78±0.59^*	38.68±0.57^**	38.63±0.51^**
水牛角SHF高剂量组	1.25	37.85±0.30	39.12±0.25^###	38.67±0.36^**	38.61±0.35^**	38.39±0.41^***
水牛角水提液组	3.125	37.67±0.27	38.90±0.25^###	38.71±0.39^*	38.61±0.51^**	38.32±0.44^***

分组	剂量/(g·kg^-1)	给药2 h	给药2.5 h	给药3.5 h	给药4.5 h
空白组		36.87±0.16	36.93±0.18	36.84±0.20	36.99±0.25
模型组		38.85±0.21^###	38.71±0.28^###	38.61±0.23^###	38.47±0.21^###
阿司匹林组	0.10	37.83±0.24^***	37.81±0.25^***	37.90±0.27^***	37.75±0.29^***
水牛角SHF低剂量组	0.312 5	38.16±0.49^***	38.14±0.43^**	38.13±0.42^*	37.94±0.44^*
水牛角SHF中剂量组	0.625	38.36±0.47^**	38.30±0.30^**	38.24±0.37^*	37.99±0.31^**
水牛角SHF高剂量组	1.25	38.20±0.30^***	38.11±0.31^**	37.94±0.28^***	37.87±0.29^**
水牛角水提液组	3.125	38.20±0.40^***	38.14±0.51^**	38.04±0.31^**	37.93±0.27^**
注：与空白组相比, ^###P < 0.001;与模型组相比, ^P < 0.05, ^{ }P < 0.01, ^{ * *}P < 0.001。

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表 4 潜在差异代谢物

Table 4. Potential differential metabolites

No.	t_R/min	生物标志物	ESI	m/z	含量差异
No.	t_R/min	生物标志物	ESI	m/z	M vs C	S vs M
1	1.78	alpha-CEHC glucuronide	+	455.190 8	↑^###	↓^***
2	10.70	PC[DiMe(9, 5)/DiMe(9, 3)]	+	839.567 1	↑^###	↓^***
3	10.72	Piritramide	+	431.276 6	↑^###	↓^***
4	13.24	TG[18∶1(11Z)/16∶1(9Z)/18∶2(9Z, 12Z)]	+	872.769 5	↑^###	↓^***
5	12.42	Trihexosylceramide (d18∶1/16∶0)	+	1041.695 1	↓^###	↑^***
6	10.72	Adrenic acid	+	355.261 7	↑^##	↓^***
7	11.69	Keratan	+	1 070.143 7	↑^###	↓^**
8	7.61	1'-O-Acetylpaxilline	+	478.266 9	↑^###	↓^**
9	13.09	15-Oleoylsolamin	+	846.758 0	↓^###	↑^*
10	11.66	PA(8∶0/13∶0)	+	512.337 7	↓^##	↑^*
11	13.24	TG[14∶0/20∶3(5Z, 8Z, 11Z)/20∶2n6]	+	898.787 8	↑^###	↓^*
12	13.62	TG[14∶0/20∶3(5Z, 8Z, 11Z)/16∶0]	+	846.757 7	↓^###	↑^*
13	12.89	N-hexadecanoylsphinganine-1-phosphocholine	+	705.585 6	↑^##	↓
14	14.10	TG[16∶0/18∶1(11Z)/18∶3(6Z, 9Z, 12Z)]	+	855.747 1	↑^###	↓
15	12.73	Ligustroside	+	525.203 8	↓^###	↑
16	15.71	PC[18∶0/22∶5(7Z, 10Z, 13Z, 16Z, 19Z)]	+	836.625 2	↓^###	↑
17	9.39	3, 4, 5-Trihydroxy-6-{[(2Z)-2-(phenylmethylidene)Heptyl]oxy}oxane-2-carboxylic acid	-	425.187 2	↑^##	↓^***
18	11.88	LysoPA[20∶4(8Z, 11Z, 14Z, 17Z)/0∶0]	-	457.238 4	↓^##	↑^**
19	15.71	all-trans-Decaprenyl diphosphate	-	857.553 6	↑^###	↓^**
20	15.28	Isomorellic acid	-	605.248 0	↑^#	↓^*
21	4.95	Xylometazoline	-	289.193 1	↓^##	↑^**
22	4.35	Hexobarbital	-	281.111 6	↓^###	↑^***
23	4.92	Busulfan	-	245.014 9	↓^###	↑^***
24	1.17	3-Dehydroquinate	-	189.040 5	↑^###	↓^**
25	15.72	PI[18∶1(11Z)/16∶0]	-	881.533 5	↑^#	↓^**
26	11.89	LysoPA[0∶0/18∶2(9Z, 12Z)]	-	433.239 1	↓^#	↑^**
27	3.92	Phenol	-	93.034 7	↑^###	↓^***
28	11.42	Valerenolic acid	-	249.150 7	↑	↓
29	13.09	PE[20∶0/16∶1(9Z)]	-	790.551 7	↓	↑
30	1.16	Uric acid	-	167.019 6	↓	↑
31	11.47	Sphingosine 1-phosphate	-	378.244 8	↑	↓
注: C.空白组; M.模型组; S.水牛角SHF组; 与空白组相比, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001;与模型组相比, ^P < 0.05, ^{ }P < 0.01, ^{ * *}P < 0.001。

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