不同方法提取白芨葡甘聚糖的初步结构特征及对心肌细胞保护作用的比较研究

doi:10.5246/jcps.2024.08.051

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (8): 686-704.DOI: 10.5246/jcps.2024.08.051

不同方法提取白芨葡甘聚糖的初步结构特征及对心肌细胞保护作用的比较研究

潘峰¹^,³, 杨楠¹, 阮国永², 喻昌燕¹, 杨建波², 锁冬雪¹, 秦颖¹, 刘云¹^,²^,³^,⁴^,^*()

^1. 遵义医科大学贵州省普通高等学校特色药物肿瘤重点防治实验室, 贵州遵义 563000
^2. 贵州光正制药有限责任公司, 贵州贵阳 550000
^3. 遵义医科大学基础医学院生物化学与分子生物学系, 贵州遵义 563000
^4. 遵义医科大学附属医院法医学鉴定中心, 贵州遵义 563000

收稿日期:2023-11-14 修回日期:2023-12-27 接受日期:2024-02-20 出版日期:2024-08-30 发布日期:2024-08-30
通讯作者: 刘云

Comparative analysis of structural characteristics and cardiomyocyte protective effects of glucomannan extracted from Bletilla striata using varied methods

Feng Pan¹^,³, Nan Yang¹, Guoyong Ruan², Changyan Yu¹, Jianbo Yang², Dongxue Suo¹, Ying Qin¹, Yun Liu¹^,²^,³^,⁴^,^*()

¹ Guizhou Provincial College-based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines, Zunyi Medical University, Zunyi 563000, Guizhou, China
² Guizhou Guangzheng Pharmaceutical Co., Ltd., Guiyang 550000, Guizhou, China
³ Department of Biochemistry and Molecular Biology, Zunyi Medical University, Zunyi 563000, Guizhou, China
⁴ Center of Forensic Expertise, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, Guizhou, China

Received:2023-11-14 Revised:2023-12-27 Accepted:2024-02-20 Online:2024-08-30 Published:2024-08-30
Contact: Yun Liu
Supported by:
Science and Technology Foundation of Guizhou Province (Grant No. QKHJC-ZK[2023]YB524); Science and Technology Plan Project of Guizhou, China (Grant No. QKPTRC[2019]035); Science and Technology Foundation of Guizhou Province (Grant No. QKH[2019]1346); Science and Technology Department of Zunyi city of Guizhou province of China (Grant No. [2020]7) and Undergraduate Training Program for Innovation and Entrepreneurship of Zunyi Medical University (Grant No. S202310661248).

摘要/Abstract

摘要：

阿霉素(DOX)广泛用于肿瘤治疗, 但是具有明显毒副作用。临床研究表明白芨可以有效防治化疗药物的毒副作用。多糖作为白芨的主要活性成分, 主要为葡甘聚糖。为了明确提取方法对获取的白芨多糖结构影响, 同时筛选出较强抗DOX心肌毒性的多糖成分, 本研究使用热水提取(WE)、超声波辅助提取(UAE)、酶辅助方法(EAE)、稀酸水提取(ACWE)和稀碱水提取(ALWE)对白芨块茎多糖组分进行了提取, 并在80%和95%的乙醇终浓度下进行醇沉, 得到16种多糖。随后, 使用称重法、苯酚硫酸法、BCA蛋白质测定法、高效凝胶渗透色谱(HPGPC)、柱前衍生高效液相色谱(HPLC)、傅里叶变换红外光谱(FT-IR)和氢核磁共振系统地分析了提取效率、总碳水化合物含量、总蛋白质含量、初步结构特性; 同时利用H9c2 (2-1) DOX损伤模型测定了不同白芨多糖抗DOX心肌细胞毒性能力。结果表明, 不同提取方法可以明显的影响白芨多糖的提取效率、总多糖含量、总蛋白含量、色素含量, 糖链结构和心肌细胞保护活性。其中, 使用WE和UAE提取BsPs的产量较高; WE或UAE、EAE和ACWE与ALWE提取BsPs的相对分子质量(RMW)分布有显著差异; 从WE和UAE获得的主要BsPs的RMW (1.9 × 10⁷–1.7 × 10⁷ Da), 高于从EAE (7.5 × 10³–2.8 × 10⁴ Da)和ALWE (5.1 × 10⁴–1.7 × 10⁴ Da); 较小的分子量主要由更高浓度的乙醇沉淀; BsPs由不同摩尔比的Man和Glu组成, 此外部分多糖提取物含有少量Gal或Ara; ACWE的BsPs显示出最大的结构差异, 它缺乏1,4-α-D-Glcp和三螺旋结构。此外, 从WE、UAE和EAE获得的BsPs对大鼠心肌细胞毒性的抗DOX活性高于其他方法。总之, 本研究强调了提取方法对BsPs结构和心肌保护活性的影响, 为探索BsPs结构的多样性和使用特定方法有效提取具有强心肌保护活性多糖提供了基础。

关键词: 白芨, 多糖, 提取方法, 结构特征, 心肌保护

Abstract:

Doxorubicin (DOX) is a widely employed tumor therapy, yet its substantial toxic side effects pose a considerable challenge. Bletilla striata has demonstrated efficacy in preventing and treating these toxic side effects in clinical practice, with polysaccharides identified as the principal active component. In the present study, 16 fractions of B. striata polysaccharides (BsPs) were extracted using diverse methods, including hot-water extraction (HWE), ultrasonic-assisted extraction (UAE), enzyme-assisted extraction (EAE), dilute acid-water extraction (ACWE), and dilute alkali-water extraction (ALWE). These extractions were subsequently precipitated at final ethanol concentrations of 80% and 95%, respectively. The investigation encompassed yields, total carbohydrate content (TCC), total protein content (TPC), preliminary structural properties, and anti-DOX myocardial cytotoxic activity. Results indicated that the extraction method significantly influenced the physicochemical properties, associated functional properties, and anti-DOX myocardial cytotoxic activity of BsPs. HWE and UAE yielded higher BsPs quantities. The relative molecular weight (RMW) distribution of BsPs differed notably between HWE or UAE, EAE, ACWE, and ALWE. The RMW of primary BsPs obtained from HWE and UAE (1.9 × 10⁷–1.7 × 10⁷ Da) exceeded that from EAE (7.5 × 10³–2.8 × 10⁴ Da) and ALWE (5.1 × 10⁴–1.7 × 10⁴ Da), with smaller molecular weights primarily precipitated by higher ethanol concentrations. BsPs were composed of Man and Glu, with partial fractions containing small amounts of Gal or Ara, displaying varying molar ratios. Notably, BsPs from ACWE exhibited the most significant structural differences, lacking 1,4-α-D-Glcp and a triple-helical structure. Furthermore, BsPs obtained from HWE, UAE, and EAE demonstrated heightened anti-DOX myocardial cytotoxic activity compared to other methods. This study underscored the influence of extraction methods on BsPs’ structure and myocardial protective activity, offering a foundation for exploring structural diversity and employing specific extraction methods to extract polysaccharides with robust myocardial protective properties efficiently.

Key words: Bletilla striata (Thunb.) Rchb. f., Polysaccharide, Extraction methods, Structural characteristics, Myocardial protection

Supporting:

潘峰, 杨楠, 阮国永, 喻昌燕, 杨建波, 锁冬雪, 秦颖, 刘云. 不同方法提取白芨葡甘聚糖的初步结构特征及对心肌细胞保护作用的比较研究[J]. 中国药学(英文版), 2024, 33(8): 686-704.

Feng Pan, Nan Yang, Guoyong Ruan, Changyan Yu, Jianbo Yang, Dongxue Suo, Ying Qin, Yun Liu. Comparative analysis of structural characteristics and cardiomyocyte protective effects of glucomannan extracted from Bletilla striata using varied methods[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(8): 686-704.

图/表 11

Figure 1. Crucial procedures related to the extraction of BsPs.

Figure 2. Analysis of yield and composition characteristics of BsPs extracted by different extraction methods. The values are the mean ± SD except a and b (n = 3). (a) The extraction rate of BsPs precipitated with 80% ethanol (BsPs-80) using different extraction methods; (b) The extraction rate of BsPs precipitated with 95% ethanol (BsPs-95) using different extraction methods; (c) Total polysaccharide content of BsPs-80 extracted by different methods; (d) Total polysaccharide content of BsPs-95 extracted by different methods; (e) TPC of BsPs-80 extracted by different methods; (f) TPC of BsPs-95 extracted by different methods; (g) The absorption intensity of BsPs-80 samples extracted by different methods at 420 nm; (h) The absorption intensity of BsPs-95 samples extracted by different methods at 420 nm. According to the ANOVA, values with the same letter are not significantly different (P < 0.05), where the letters include a to f.

Figure 3. High-performance gel permeation chromatograms of BsPs extracted by different extraction methods.

Table 1. The RMW of BsPs calculated based on their RT values obtained from high-performance gel permeation chromatograms.

Figure 4. FT-IR spectrums of BsPs extracted by different extraction methods.

Figure 5. HPLC-DAD chromatograms of monosaccharide standards and BsPs extracted by different extraction methods.

Table 2. The molar ratio of monosaccharide composition for BsPs extracted by different extraction methods.

Figure 6. 1H NMR spectrum of BsPs extracted by different extraction methods.

Figure 7. The maximum absorption wavelengths of BsPs-Congo red complexes over the NaOH concentration range of 0–0.5 M.

Figure 8. Cell morphology observation of rat cardiomyocytes H9c2 (2-1) after SRB staining with the treatment of BsPs obtained by different extraction methods in combination with DOX.

Figure 9. The cell survival rate of rat cardiomyocytes H9c2 (2-1) after SRB staining with the treatment of BsPs obtained by different extraction methods in combination with DOX. According to the ANOVA, values with the same letter are not significantly different (P < 0.05), where the letters include a to e.

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不同方法提取白芨葡甘聚糖的初步结构特征及对心肌细胞保护作用的比较研究

Comparative analysis of structural characteristics and cardiomyocyte protective effects of glucomannan extracted from Bletilla striata using varied methods

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