蓝靛果多糖的分离纯化及免疫调节活性研究

doi:10.12122/j.issn.1673-4254.2026.08.08

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (8): 1799-1813.doi: 10.12122/j.issn.1673-4254.2026.08.08

• • 上一篇

蓝靛果多糖的分离纯化及免疫调节活性研究

黄旸家¹(), 张旭东²(), 向倩如¹^,³, 郭汉权¹, 郑爽¹, 郝宇婷⁴, 廖文镇¹()

^1.南方医科大学公共卫生学院营养与食品卫生学系，广东省热带病研究重点实验室，广东广州 510515
^2.深圳市坪山区疾病预防控制中心，广东深圳 518118
^3.南方医科大学珠江医院第二临床医学院临床营养科，广东广州 510280
^4.广州市老年医院感染预防控制科，广东广州 510030

收稿日期:2026-01-23 出版日期:2026-08-20 发布日期:2026-08-01
通讯作者: 廖文镇 E-mail:2398723665@qq.com;841528342@qq.com;wenzhenliao@163.com
作者简介:黄旸家，在读硕士研究生，E-mail: 2398723665@qq.com
张旭东，硕士，医师，E-mail: 841528342@qq.com。
第一联系人：黄旸家、张旭东共同为第一作者
基金资助:
广东省自然科学基金(2025A1515010492);广东省自然科学基金(2023A1515011065)

Isolation, purification and immunomodulatory activity of polysaccharide from Lonicera caerulea L. var. edulis Turcz. ex Herder

Yangjia HUANG¹(), Xudong ZHANG²(), Qianru XIANG¹^,³, Hanquan GUO¹, Shuang ZHENG¹, Yuting HAO⁴, Wenzhen LIAO¹()

^1.Department of Nutrition and Food Hygiene, School of Public Health, Southern Medical University, Guangdong Key Laboratory of Tropical Disease Research, Guangzhou 510515, China
^2.Shenzhen Pingshan District Center for Disease Control and Prevention, Shenzhen 518118, China
^3.Department of Clinical Nutrition, Second Clinical Medical College, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China
^4.Department of Infection Prevention and Control, Guangzhou Geriatric Hospital, Guangzhou 510030, China

Received:2026-01-23 Online:2026-08-20 Published:2026-08-01
Contact: Wenzhen LIAO E-mail:2398723665@qq.com;841528342@qq.com;wenzhenliao@163.com

摘要/Abstract

摘要：

目的旨在从蓝靛果（L. caerulea）中分离提取出一种新型纯化蓝靛果多糖（THP-2），探究其免疫调节活性。方法采用超声辅助水提醇沉法和凝胶柱层析等技术对L. caerulea进行多糖分离提取与纯化工艺，通过凝胶渗透色谱、离子色谱、高碘酸氧化与smith降解、红外光谱、核磁共振波谱以及刚果红实验等多组学方法，系统表征THP-2的相对分子质量、纯度及化学结构进行表征和分析。体外细胞实验采用DC2.4细胞和Raw264.7细胞模型对THP-2免疫调节活性进行初步评价。结果本研究从L. caerulea中分离纯化出一种新型多糖组分THP-2，其化学组成和结构特征与既往文献报道的蓝靛果多糖（THP）存在显著差异。该多糖的平均相对分子质量为123 090，单糖组分包括糖、半乳糖、葡萄糖、甘露糖、木糖、葡萄糖醛酸和半乳糖醛酸，摩尔比为27.84∶52.28∶8.47∶6.05∶2.75∶0.90∶0.88∶0.83。糖苷键分析表明，THP-2主要由（1→5）、（1→3）、（1→4）、（1→6）、（1→3，4）、（1→6，4）等糖苷键组成。免疫活性研究表明，THP-2能够显著激活DC2.4细胞的共刺激分子CD80、CD86、CD40、MHC-Ⅱ的表达（P<0.05），促进促炎因子IL-1β、IL-6、TNF-α的分泌（P<0.05），同时上调Raw264.7细胞的共刺激分子CD80、CD86、CD40的表达（P<0.05），增强促炎因子IL-1β、IL-6、TNF-α表达（P<0.05）。此外，THP-2还能促进DC2.4细胞与Raw264.7细胞NO合成相关分子iNOS表达（P<0.05），增加NO分泌量（P<0.05），上调胞内促炎相关蛋白p-p65、p-IκB表达水平（P<0.05），增加DC2.4细胞的p-Akt蛋白含量（P<0.05）。结论本研究成功从L. caerulea中分离纯化出一种新型多糖组分THP-2，并对其化学结构进行了系统鉴定和表征。THP-2主要通过激活NF-κB信号通路和PI3K/Akt通路促进DC2.4细胞成熟分化，并通过NF-κB通路促进Raw264.7细胞向M1型巨噬细胞分化的能力。

关键词: 蓝靛果, 多糖, 化学结构, 免疫调节, 细胞极化

Abstract:

Objective To isolate a novel polysaccharide (THP-2) from Lonicera caerulea L.var. edulis Turcz. ex Herder (L. caerulea) and evaluate its immunomodulatory activity. Methods Ultrasonic-assisted water extraction and alcohol precipitation and gel column chromatography were used to separate and purify polysaccharides from L.caerulea. The relative molecular mass, purity and chemical structure of THP-2 were analyzed by gel permeation chromatography, ion chromatography, periodate oxidation and smith degradation, infrared spectroscopy, nuclear magnetic resonance spectroscopy and Congo red experiment. DC2.4 cells and Raw264.7 cells were used to evaluate the immunomodulatory activity of THP-2. Results A new polysaccharide component THP-2 was isolated and purified from L.caerulea, and its chemical composition and structural characteristics were significantly different from those of L.caerulea polysaccharide reported previously. The average relative molecular mass of THP-2 was 123 090, and its monosaccharide components included sugar, galactose, glucose, mannose, xylose, glucuronic acid and galacturonic acid with a molar ratio of 27.84:52.28:8.47:6.05:2.75:0.90:0.88:0.83. Glycosidic bond analysis showed that THP-2 was mainly composed of (1→5), (1→3), (1→4), (1→6), (1→3,4), (1→6,4) and other glycosidic bonds. THP-2 could activate expressions of costimulatory molecules CD80, CD86, CD40 and MHC-II and promote IL-1β, IL-6 and TNF-α secretion in DC2.4 cells, up-regulate CD80, CD86 and CD40 expressions and enhance the expressions of IL-1β, IL-6 and TNF-α in Raw264.7 cells. THP-2 also promoted iNOS expression, increased NO secretion, and up-regulated p-p65 and p-IκB expressions in both cells, and increased p-Akt protein content in DC2.4 cells. Conclusion The new polysaccharide component THP-2 isolated from L.caerulea is capable of promoting maturation and differentiation of DC2.4 cells by activating the NF‑κB signaling pathway and PI3K/Akt pathway and promoting the differentiation of Raw264.7 cells into M1 macrophages through the NF-κB pathway.

Key words: Lonicera caerulea L. var. edulis Turcz. ex Herder, polysaccharide, chemical structure, immune regulation, cell polarization

黄旸家, 张旭东, 向倩如, 郭汉权, 郑爽, 郝宇婷, 廖文镇. 蓝靛果多糖的分离纯化及免疫调节活性研究[J]. 南方医科大学学报, 2026, 46(8): 1799-1813.

Yangjia HUANG, Xudong ZHANG, Qianru XIANG, Hanquan GUO, Shuang ZHENG, Yuting HAO, Wenzhen LIAO. Isolation, purification and immunomodulatory activity of polysaccharide from Lonicera caerulea L. var. edulis Turcz. ex Herder[J]. Journal of Southern Medical University, 2026, 46(8): 1799-1813.

图/表 14

表1 目的基因的引物序列

Tab.1 Primer sequences for qRT-PCR of the target genes

Gene	Primer sequence 5'-3'
GAPDH	F:AGGTCGGTGTGAACGGATTTG
GAPDH	R:GGGGTCGTTGATGGCAACA
CD80	F:TGCTGCTGATTCGTCTTTCAC
CD80	R:GAGGAGAGTTGTAACGGCAAG
CD86	F:TCAATGGGACTGCATATCTGCC
CD86	R:GCCAAAATACTACCAGCTCACT
CD40	F:GCAGTGTGTTACGTGCAGTG
CD40	R:TGTGCAGTGGCTTGTCAGTC
IL-1β	F:GCAACTGTTCCTGAACTCAACT
IL-1β	R:ATCTTTTGGGGTCCGTCAACT
IL-6	F:TAGTCCTTCCTACCCCAATTTCC
IL-6	R:TTGGTCCTTAGCCACTCCTTC
TNF- α	F:CCCTCACACTCAGATCATCTTCT
TNF- α	R:GCTACGACGTGGGCTACAG
iNOS	F:GTTCTCAGCCCAACAATACAAGA
iNOS	R:GTGGACGGGTCGATGTCAC

图1 L. caerulea粗多糖的分离纯化

Fig.1 Isolation and purification of crude polysaccharides from L. caerulea. A: Elution curve of THP by DEAE-sepharose-FF gel chromatography. B: Sephadex G-100 gel chromatography elution curve of THP-2. C: HPGPC results of THP-2. D: Standard curve of glucose. E: Standard curve of BCA protein.

图2 THP-2三氟乙酸水解产物的离子色谱图

Fig.2 Ion chromatogram of THP-2 trifluoroacetic acid hydrolysate.

图3 THP-2的高碘酸氧化和Smith降解结果

Fig.3 Results of periodate oxidation and Smith degradation of THP-2. A: THP-2 sodium periodate concentration standard curve. B: Concentration change curve of sodium periodate in the reaction. C: Gas chromatography of Smith degradation products of THP-2 (a: ethylene glycol; b: glycerol; c: erythritol; d: arabinose; e: mannose; f: inositol; g: galactose).

图4 THP-2的构象、纯度及官能团特征分析

Fig.4 Analysis of conformation, purity and functional group characteristics of THP-2. A: Congo red test results of THP-2. B: UV spectrum scan of crude polysaccharide. C: UV spectrum of THP-2. D: Infrared spectrum of THP-2.

图5 THP-2的一维核磁H谱和13C谱

Fig.5 One-dimensional 1H (A) and 13C NMR spectrum (B) of THP-2.

表2 THP-2糖残基的1H和¹³C NMR谱的化学位移

Tab.2 Chemical shifts in 1H and ¹³C NMR spectra of THP-2 sugar residues

Sugar residue	Chemical shift (ppm)
Sugar residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
5)-α-L-Alaf(1→	109.23/5.25	80.1/4.45	76.56/3.93	82.21/4.23	-/-	-/-
3)-α-L-Alaf(1→	107.49/5.16	76.56/-	81.32/3.72	82.32/4.14	61.03/-	/-
4)-β-D-Galp(1→	104.35/4.55	71.8/3.56	73.29/3.7	77.65/4.14	/-	60.73/3.73
3,4)-β-D-Xylp(1→	103.68/4.47	74.5/3.28	79.82/3.55	74.96/-	63.28/-	0/-
6)-β-D-Glup(1→	103.4/4.52	71.8/3.55	76.56/3.31	69.85/3.54	75.13/3.66	69.47/3.83
4,6)-β-D-Glup(1→	103.13/4.5	73.38/3.31	76.56/-	70.71/-	72.96/-	69.47/4.23
4)-β-D-Glup(1→	102.6/4.54	72.58/3.36	83.84/3.54	68.44/3.29	-/3.31	61.26/3.83
4)-β-D-Manp(1→	101.63/4.66	71.46/4.05	72.74/3.72	77.65/3.73	76.56/3.49	61.26/3.83
4,6)-β-D-Manp(1→	100.13/4.63	70.71/3.95	71.8/3.7	76.71/3.71	73.72/3.66	66.85/3.85
6)-α-D-Glup(1→	99.36/5.12	72.18/3.95	74.5/4.05	69.85/3.85	70.71/3.93	66.85/-

图6 THP-2对DC2.4 细胞增殖能力的影响

Fig.6 Effect of THP-2 on proliferation of DC2.4 cells. **P<0.01.

图7 THP-2对DC2.4细胞共刺激分子CD80 (A)、CD40 (B)、CD86 (C)、MHC-II (D)表达情况及其MFI值的影响

Fig.7 Effects of THP-2 on expressions of the costimulatory molecules CD80 (A), CD40 (B), CD86 (C), MHC-II (D) and their MFI values in DC2.4 cells. *P<0.05, **P<0.01 vs THP-2 (0 μg/mL) + LPS (0 μg/mL).

图7 THP-2对DC2.4细胞共刺激分子CD80 (A)、CD40 (B)、CD86 (C)、MHC-II (D)表达情况及其MFI值的影响

图8 THP-2对DC2.4细胞qPCR与ELISA实验结果

Fig.8 qPCR and ELISA results of THP-2 on DC2.4 cells. A: Relative expression of CD80 mRNA. B: Relative expression of CD86 mRNA. C: Relative expression of CD40 mRNA. D: Relative expression of TNF-α mRNA. E: Relative expression of IL-1β mRNA. F: Relative expression of iNOS mRNA. G: Relative expression of IL-6 mRNA. H: IL-6 content in culture supernatant. I: Content of NO in the culture supernatant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs THP-2 (0 μg/mL)+LPS (0 μg/mL).

图9 THP-2对Raw264.7细胞增殖能力的影响

Fig.9 Effect of THP-2 on proliferation of Raw264.7 cells. ***P<0.001, ****P<0.0001 vs THP-2 (0 μg/mL).

图10 THP-2对Raw264.7细胞qPCR与ELISA实验结果

Fig.10 qPCR and ELISA results of THP-2 on Raw264.7 cells. A: Relative expression of CD80 mRNA. B: Relative expression of CD86 mRNA. C: Relative expression of CD40 mRNA. D: Relative expression of TNF-α mRNA. E: Relative expression of IL-1β mRNA. F: Relative expression of iNOS mRNA. G: Content of TNF-α in culture supernatant. H: Content of IL-6 in culture supernatant. I: Content of NO in the culture supernatant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs THP-2 (0 μg/mL)+LPS (0 μg/mL).

图11 THP-2对DC2.4细胞p-p65、p-lκB、p-Akt蛋白含量的影响以及对Raw264.7细胞p-IκB、p-p65蛋白含量的影响

Fig.11 Effect of THP-2 on p-p65, p-lκB, and p-Akt protein expressions in DC2.4 cells and p-IκB and p-p65 protein expressions in Raw264.7 cells. A: Content of p-p65 protein in DC2.4 cells. B: Content of p-IκB protein in DC2.4 cells. C: Content of p-Akt protein in DC2.4 cells. D: Content of p-IκB protein in Raw264.7 cells. E: Content of p-p65 protein in Raw264.7 cells. *P<0.05, **P<0.01, ****P<0.0001 vs THP-2 (0 μg/mL)+LPS (0 μg/mL).

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蓝靛果多糖的分离纯化及免疫调节活性研究

Isolation, purification and immunomodulatory activity of polysaccharide from Lonicera caerulea L. var. edulis Turcz. ex Herder

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