药学学报

Source	Isolation method	Size/nm	Function	Constituent	Ref.
Aqueous extract of Turkish gall	Differential centrifugation	Size decreased with the increasing centrifugal force, 6 577 (×g)-9 500 (×g) TG-LP NPs: 413.89 ± 202.95 TG-HP NPs: 230.34 ± 59.48	Potent pH responsiveness, free radical scavenging, and antibacterial effects	Galli cactechol oligomers	[24]
Black tea infusion	SEC	187 ± 7	Antioxidant activity	Protein, polysaccharides, caffeine, gallic acid and catechins	[25-27]
Freshwater clam (Corbicula fluminea Muller) soup	Centrifugation, SEC-DLS	Clam soup: 74.82 ± 0.68; fractions isolated by SEC F1: 70.86 ± 1.82 F2: 45.88 ± 4.16 F3: 50.35 ± 9.67	Hypolipidemic effect and improve nonalcoholic fatty liver disease	Taurine, ornithine and phytosterols; protein, carbohydrate and lipid	[10, 28]
Centrifugation, ultrafiltration	Clam soup: 77.52 ± 1.50
Aqueous extract of Armeniacae Semen Amarum	Centrifugation, dialysis, anion exchange and gel filtration chromatography, thermal induction	92	Encapsulate paclitaxel	Polypeptides	[13]
Coptidis Rhizoma decoction	Ultra-centrifugation and anion exchange chromatography	Fractions obtained by elution: CCD-P1: 272.7 ± 25.2 CCD-P2: 264.8 ± 21.4 CCD-P3: 427.4 ± 41.8	Promote intestinal berberine absorption	Mannose, rhamnose, glucose, galactose, arabinose and other sugars; galacturonic acid and little protein	[15]
Centrifugation, alcohol precipitation and Sevage method	Size increased with the increase of concentration (20-80 μg·mL^-1) CCP-P1: ~200 CCP-P2: ~380 CCP-P3: ~415	Promote intestinal berberine absorption; inhibit advanced glycation end product formation	[15, 29]
Aqueous extract of Glycyrrhizae Radix et Rhizoma	Precipitation with 50% ethanol-water solution, purified with anionic exchange chromatography followed by hydrophobic interaction chromatography, thermal induction	74.1 ± 0.7	Promote cellular uptake of Caco-2, L02, Hep-G2, macrophage NR8383 and proliferation of normal hepatocytes; solubilize the insoluble astragaloside IV by encapsulation	Protein	[11]
Isatidis Radix decoction	SEC-MALLS	~120	Promote the growth of normal cells but suppress that of cancerogenic cells and macrophages	Protein	[14]
Centrifugation, SEC-DLS	57-300	Antiviral effects	Polysaccharides	[16]
Puerariae thomsonii Radix decoction	Preparation of different concentrations of Fenge water decoction	Size increased with the increase of concentration (0.07-0.35 g·mL^-1) 0.07 g·mL^-1 (F-1): 120 0.10 g·mL^-1 (F-2): 195 0.17 g·mL^-1 (F-3): 256 0.35 g·mL^-1 (F-4): 480	Promote the absorption of ingredients in decoction and reduce AngⅡ level in acute blood stasis model rats	-	[30]
Aqueous extract of Rabdosia rubescentis leaves	Centrifugation	Size increased with the decrease of concentration (1.0-0.12 mg·mL^-1) 4.5-234.3	Inhibit thrombosis and antiplatelet aggregation	Nucleotides, diterpenoids, rosmarinic acid and other phenolic acids	[31]
Green tea infusion	Centrifugation, dialysis, SEC-HPLC	100-300	Immunostimulatory effect; load doxorubicin for drug delivery	Protein, polysaccharides	[32]
Puerariae lobatae Radix decoction	Ultra-centrifugation and filtration	200-351	Improve tiny circulation	Puerarin, daidzein, daidzin, genistein	[33]
Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma decoction	Water extract and acetone precipitation	185.5	Antibacterial activity	Protein, berberine	[34]
Glycyrrhizae Radix et Rhizoma and Aconiti lateralis Radix Praeparata decoction	Ethanol precipitation (40%-60%), anionic exchange chromatography and hydrophobic chromatography, adjust pH to 5 with HCl	Licorice root protein nanoparticles (GP NPs): 206.20 ± 2.02; GP-aconitine nanoparticles (GP-AC NPs): 238.20 ± 1.23	Reduce the toxicity of aconitine	Protein, aconitine	[35]
Naoluo Xintong decoction	Differential centrifugation and dialysis	200	Play an essential role in the brain-protective effect of decoction	Ginsenoside Rg₁, ginsenoside Rb₁, astragaloside IV, protein, polysaccharides	[36]
Shaoyao Gancao decoction	Ultra-centrifugation	200	Encapsulate the components of Baishao and facilitate the absorption of drugs in the ileum	Albiflorin, paeoniflorin and benzoylpaeoniflorin	[37, 38]
Shengmai Yin (pulse-activating decoction)	Centrifugation and dialysis	Decoction: 450.80 ± 113.61; colloidal phase: 87.53 ± 1.57; suspension phase: 2 938.67 ± 168.15	Alleviate the symptoms of heart failure in SD rats with deficiency of both qi and yin	-	[39]
Baihu decoction	Differential centrifugation and dialysis	100	Antipyretic effect	Neomangiferin, mangiferin, glycyrrhizic acid and ammonium glycyrrhizinate; C, O, Mg, Al, Si, Ca, Zn and other elements	[40, 41]
Ge-Gen-Qin-Lian decoction	Ultra-centrifugation	Microaggregates (MA): 2 775 ± 712; micro/nanoaggregates (MNA): 531 ± 23	Antioxidant cellular protection and improve the bioavailability of baicalin	-	[42]
Ma-Xing-Shi-Gan decoction	Centrifugation, SEC-MALLS	50-150	Influence cell viability	Ephedrine, pseudoephedrine	[43]
Centrifugation	S-13500: 170	Antibacterial activity	Ephedrine, amygdalin, glycyrrhizic acid and inorganic components Ca, K, Mg, etc	[44]

Source	Isolation method	Size/nm	Function	Constituent	Ref.
Aqueous extract of Turkish gall	Differential centrifugation	Size decreased with the increasing centrifugal force, 6 577 (×g)-9 500 (×g) TG-LP NPs: 413.89 ± 202.95 TG-HP NPs: 230.34 ± 59.48	Potent pH responsiveness, free radical scavenging, and antibacterial effects	Galli cactechol oligomers	[24]
Black tea infusion	SEC	187 ± 7	Antioxidant activity	Protein, polysaccharides, caffeine, gallic acid and catechins	[25-27]
Freshwater clam (Corbicula fluminea Muller) soup	Centrifugation, SEC-DLS	Clam soup: 74.82 ± 0.68; fractions isolated by SEC F1: 70.86 ± 1.82 F2: 45.88 ± 4.16 F3: 50.35 ± 9.67	Hypolipidemic effect and improve nonalcoholic fatty liver disease	Taurine, ornithine and phytosterols; protein, carbohydrate and lipid	[10, 28]
Centrifugation, ultrafiltration	Clam soup: 77.52 ± 1.50
Aqueous extract of Armeniacae Semen Amarum	Centrifugation, dialysis, anion exchange and gel filtration chromatography, thermal induction	92	Encapsulate paclitaxel	Polypeptides	[13]
Coptidis Rhizoma decoction	Ultra-centrifugation and anion exchange chromatography	Fractions obtained by elution: CCD-P1: 272.7 ± 25.2 CCD-P2: 264.8 ± 21.4 CCD-P3: 427.4 ± 41.8	Promote intestinal berberine absorption	Mannose, rhamnose, glucose, galactose, arabinose and other sugars; galacturonic acid and little protein	[15]
Centrifugation, alcohol precipitation and Sevage method	Size increased with the increase of concentration (20-80 μg·mL^-1) CCP-P1: ~200 CCP-P2: ~380 CCP-P3: ~415	Promote intestinal berberine absorption; inhibit advanced glycation end product formation	[15, 29]
Aqueous extract of Glycyrrhizae Radix et Rhizoma	Precipitation with 50% ethanol-water solution, purified with anionic exchange chromatography followed by hydrophobic interaction chromatography, thermal induction	74.1 ± 0.7	Promote cellular uptake of Caco-2, L02, Hep-G2, macrophage NR8383 and proliferation of normal hepatocytes; solubilize the insoluble astragaloside IV by encapsulation	Protein	[11]
Isatidis Radix decoction	SEC-MALLS	~120	Promote the growth of normal cells but suppress that of cancerogenic cells and macrophages	Protein	[14]
Centrifugation, SEC-DLS	57-300	Antiviral effects	Polysaccharides	[16]
Puerariae thomsonii Radix decoction	Preparation of different concentrations of Fenge water decoction	Size increased with the increase of concentration (0.07-0.35 g·mL^-1) 0.07 g·mL^-1 (F-1): 120 0.10 g·mL^-1 (F-2): 195 0.17 g·mL^-1 (F-3): 256 0.35 g·mL^-1 (F-4): 480	Promote the absorption of ingredients in decoction and reduce AngⅡ level in acute blood stasis model rats	-	[30]
Aqueous extract of Rabdosia rubescentis leaves	Centrifugation	Size increased with the decrease of concentration (1.0-0.12 mg·mL^-1) 4.5-234.3	Inhibit thrombosis and antiplatelet aggregation	Nucleotides, diterpenoids, rosmarinic acid and other phenolic acids	[31]
Green tea infusion	Centrifugation, dialysis, SEC-HPLC	100-300	Immunostimulatory effect; load doxorubicin for drug delivery	Protein, polysaccharides	[32]
Puerariae lobatae Radix decoction	Ultra-centrifugation and filtration	200-351	Improve tiny circulation	Puerarin, daidzein, daidzin, genistein	[33]
Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma decoction	Water extract and acetone precipitation	185.5	Antibacterial activity	Protein, berberine	[34]
Glycyrrhizae Radix et Rhizoma and Aconiti lateralis Radix Praeparata decoction	Ethanol precipitation (40%-60%), anionic exchange chromatography and hydrophobic chromatography, adjust pH to 5 with HCl	Licorice root protein nanoparticles (GP NPs): 206.20 ± 2.02; GP-aconitine nanoparticles (GP-AC NPs): 238.20 ± 1.23	Reduce the toxicity of aconitine	Protein, aconitine	[35]
Naoluo Xintong decoction	Differential centrifugation and dialysis	200	Play an essential role in the brain-protective effect of decoction	Ginsenoside Rg₁, ginsenoside Rb₁, astragaloside IV, protein, polysaccharides	[36]
Shaoyao Gancao decoction	Ultra-centrifugation	200	Encapsulate the components of Baishao and facilitate the absorption of drugs in the ileum	Albiflorin, paeoniflorin and benzoylpaeoniflorin	[37, 38]
Shengmai Yin (pulse-activating decoction)	Centrifugation and dialysis	Decoction: 450.80 ± 113.61; colloidal phase: 87.53 ± 1.57; suspension phase: 2 938.67 ± 168.15	Alleviate the symptoms of heart failure in SD rats with deficiency of both qi and yin	-	[39]
Baihu decoction	Differential centrifugation and dialysis	100	Antipyretic effect	Neomangiferin, mangiferin, glycyrrhizic acid and ammonium glycyrrhizinate; C, O, Mg, Al, Si, Ca, Zn and other elements	[40, 41]
Ge-Gen-Qin-Lian decoction	Ultra-centrifugation	Microaggregates (MA): 2 775 ± 712; micro/nanoaggregates (MNA): 531 ± 23	Antioxidant cellular protection and improve the bioavailability of baicalin	-	[42]
Ma-Xing-Shi-Gan decoction	Centrifugation, SEC-MALLS	50-150	Influence cell viability	Ephedrine, pseudoephedrine	[43]
Centrifugation	S-13500: 170	Antibacterial activity	Ephedrine, amygdalin, glycyrrhizic acid and inorganic components Ca, K, Mg, etc	[44]

中药水煎液自组装聚集体研究面临的问题初探

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王琪 ¹^,² , 郭小萌 ¹^,² , 倪乾坤 ³^,⁴ , 李美景 ¹^,² , 徐睿 ¹^,² , 梁兴杰 ³^,⁴^,^* , 龚慕辛 ¹^,²^,^*

药学学报 | 综述 2024,59(1): 94-104

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药学学报 | 综述 2024, 59(1): 94-104

中药水煎液自组装聚集体研究面临的问题初探

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王琪¹^,², 郭小萌¹^,², 倪乾坤³^,⁴, 李美景¹^,², 徐睿¹^,², 梁兴杰³^,⁴^,^*, 龚慕辛¹^,²^,^*

作者信息

1.首都医科大学中医药学院, 北京 100069

2.中医络病研究北京市重点实验室, 北京 100069

3.国家纳米科学中心, 中国科学院纳米科学卓越创新中心, 中国科学院纳米生物效应与安全性重点实验室, 北京 100190

4.中国科学院大学, 北京 100086

通讯作者:

^*梁兴杰, E-mail: liangxj@nanoctr.cn;

龚慕辛, E-mail: gongmuxin@126.com

Challenges in the study of self-assembled aggregates in decoction of traditional Chinese medicine: A preliminary review

Qi WANG¹^,², Xiao-meng GUO¹^,², Qian-kun NI³^,⁴, Mei-jing LI¹^,², Rui XU¹^,², Xing-jie LIANG³^,⁴^,^*, Mu-xin GONG¹^,²^,^*

Affiliations

1. School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China

2. Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Beijing 100069, China

3. Chinese Academy of Sciences (CAS) Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190, China

4. University of Chinese Academy of Sciences, Beijing 100086, China

出版时间: 2024-01-12 doi: 10.16438/j.0513-4870.2023-0356

文章导航

摘要

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汤剂是中医临床用药的常用剂型。中药水煎液自组装聚集体(self-assembled aggregates in decoction, SADs) 是指在煎煮过程中, 中药活性成分受热运动剧烈, 在氢键、π-π堆积、疏水相互作用、静电作用等分子作用力下自组装形成的颗粒、凝胶、纤维等聚集体。研究发现SADs普遍存在于中药水煎液中, 并可发挥优于有效单体或其物理混合物的生物活性, 从成分互作-相态结构角度为揭示中药药效物质基础提供新的思路, 现已成为研究热点。本文总结归纳近年相关研究, 发现水煎液体系的多分散性、煎煮过程中活性成分相互作用的不稳定性、聚集体自组装规律的未知性、产品物理化学稳定性、纯度与产率等问题在研究中值得关注, 并提出解决思路和未来研究展望, 以期为SADs的综合开发和临床应用提供参考。

关键词

自组装 / 汤剂 / 相态 / 聚集 / 药效物质

Abstract

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Decoction is the most commonly used dosage form in the clinical treatment of traditional Chinese medicine (TCM). During boiling, the violent movement of various active ingredients in TCM creates molecular forces such as hydrogen bonding, π-π stacking, hydrophobic interactions and electrostatic interactions, which results in the formation of self-assembled aggregates in decoction (SADs), including particles, gels, fibers, etc. It was found that SADs widely existed in decoction with biological activities superior to both effective monomers and their physical mixtures, providing a new idea to reveal the pharmacodynamic material basis of Chinese herbal medicine from the perspective of component interactions-phase structure. Recently, SADs have become a novel focus of research in TCM. This paper reviewed their relevant studies in recent years and found some issues to be concerned in the research, such as the polydispersity of decoction system, instability of active ingredient interactions during boiling, uncertainty of the aggregates self-assembly rules, and stability, purity, yield of the products. In this regard, some solutions and new ideas were presented for the integrated development and clinical application of SADs.

Key words

self-assembly / decoction / phase state / aggregation / pharmacodynamic substance

引用本文

王琪, 郭小萌, 倪乾坤, 李美景, 徐睿, 梁兴杰, 龚慕辛. 中药水煎液自组装聚集体研究面临的问题初探. 药学学报, 2024 , 59 (1) : 94 -104 . DOI: 10.16438/j.0513-4870.2023-0356

Qi WANG, Xiao-meng GUO, Qian-kun NI, Mei-jing LI, Rui XU, Xing-jie LIANG, Mu-xin GONG. Challenges in the study of self-assembled aggregates in decoction of traditional Chinese medicine: A preliminary review[J]. Acta Pharmaceutica Sinica, 2024 , 59 (1) : 94 -104 . DOI: 10.16438/j.0513-4870.2023-0356

正文

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中药汤剂应用历史悠久, 疗效显著, 以“汤者, 荡也, 煎成清汁是也, 去大病用之”展现其对病邪的扫荡之功与见效快捷。近年来, 不少研究发现中药水煎液中普遍存在自组装聚集体(self-assembled aggregates in decoction, SADs)^{[1, 2]}, 不仅可发挥抗炎、镇痛、抗菌等生物活性, 也可作为天然载体用于药物递送, 提高药物的生物利用度, 对药效发挥起到重要作用, 具有广泛的应用前景。自组装是指分子在非共价键的弱相互作用和协同效应下, 自发地形成有序结构的过程^[3]。煎煮过程中, 中药活性成分运动剧烈, 在非共价键作用力或其他因素影响下自组装形成颗粒、凝胶、纤维等聚集体, 可发挥克服药味偏性、增效减毒等作用。对于SADs的研究和关注, 弥补了传统单一的化学视角在认识和阐释中药药效物质基础研究中的局限性, 从成分互作-相态结构角度对汤液起效机制进行阐释, 证明了中药药效不仅取决于化学成分, 还与其组分间的相互作用、形成的相态结构相关, 为揭示中药药效物质基础、作用机制与阐明复方配伍科学内涵提供新思路^[4]。

然而, 中药组成成分来源广泛、结构复杂, 其水煎液是由真溶液、胶体溶液、混悬液等不同分散体系组成的混合相态体系^[5], 煎煮过程中活性成分间相互作用受多种因素影响, 加之体系的多分散性决定了其形成的聚集体粒径不均一、形貌多样; 同时由于活性分子间的自组装主要基于非共价作用力, 使得SADs往往存在物理化学稳定性较差的缺陷, 制约其进一步发展。此外, 较之中药水煎液, SADs目前相对复杂的分离纯化技术和高生产成本也值得研究者进一步思考解决。本文在梳理近年有关SADs研究的基础上, 就上述问题提出解决思路和未来展望。

1 中药水煎液自组装聚集体

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受高通量筛选技术中小分子有机化合物的聚集现象^[1]和中药复方水煎液自沉淀^[6]现象的启发, 有学者提出了“结构中药学”理论^[4], 认为传统单一的化学视角在认识和阐释中药药效物质基础方面存在局限性, 中药起效离不开活性成分适宜的存在形式。共存活性物质间的相互作用可能是中药水煎液可在某种程度上表现出明显优于纯化后单体化合物有效性和安全性的原因^[7]。相态包含成分和结构两个层面, “结构中药学”认为成分是效用的来源和基础, 结构是起效的形式和途径, 成分和结构共同影响药效的发挥^[4], 应结合化学成分和物理结构两个层面研究中药成分的相态变化规律与其效应之间的关系。有研究发现黄芩与黄连的水提液均为澄清透明液体, 然而当两种水提液被混合后, 就会出现明显且大量的沉淀, 后期研究表明, 黄芩中的活性成分黄芩苷、汉黄芩苷与黄连中的活性成分小檗碱可分别自组装形成纳米粒和纳米纤维, 且黄芩苷-小檗碱自组装纳米粒可发挥出优于小檗碱自身的抗菌效果^[8]。除植物药外, 动物药水煎液中自组装聚集体相关研究也有涉及^{[9, 10]}。

中药水煎液是一个多组分的复杂系统, 在煎煮时可产生大量多糖、蛋白质、核酸、脂类等初级代谢产物和次级代谢产物, 活性化合物在加热过程中可发生多种物理相互作用(离子键、氢键、配位键、范德华力、π-π堆积、静电作用、疏水相互作用等) 和化学反应(美拉德反应、氧化反应、水解反应、降解反应、聚合反应等), 自组装形成纳米到微米等粒径不同的聚集体^[2]。如加热可诱导蛋白质三级结构的展开和二级结构的构象变化, 随后一些特定区域(如疏水位点或游离-SH基团) 暴露出来, 导致新的分子间相互作用和聚集体的形成^[11]。

近年来, 大分子物质如太子参蛋白^[12]、苦杏仁蛋白^[13]、板蓝根蛋白^[14]、甘草蛋白^[11]等蛋白质以及黄连多糖^[15]、板蓝根多糖^[16]、当归多糖^[17]等多糖类成分自组装形成聚集体的研究被大量报道, 其可自发形成规则的聚集体, 包裹水不溶性分子, 增加难溶性成分的分散程度和生物利用度, 或可降低毒性成分的毒性, 最终起到高效吸收、降低毒性等作用。此外, 中药中的一些活性小分子如皂苷^{[18, 19]}、萜类^{[20, 21]}、生物碱^{[8, 22]}等成分亦可在煎煮时自组装形成聚集体, 其较之单体成分存在特有的优势。如甘草蛋白-乌头碱聚集体可包封有毒生物碱, 起到降低毒性的作用, 小鼠注射甘草蛋白-乌头碱自组装纳米粒后仅导致轻微的可恢复毒性, 且无死亡现象, 而注射乌头碱或无纳米粒的甘草蛋白-乌头碱简单混合物后产生严重毒性反应, 且全部死亡^[23]。中药水煎液在煎煮过程中形成的自组装聚集体及其组成成分和药理活性逐渐引起人们关注, 近期研究发现的SADs见表 1^{[10, 11, 13-16, 24-44]}。

与现代纳米技术有目的地将单分子化合物采用一定技术手段结合不同, SADs是中药煎煮过程中活性成分在非共价键等作用力下自发形成的, 这种由多种成分组装而成的纳米结构较之单一成分组装的纳米结构更具多功能性, 同时由于SADs源于中药水煎液, 与合成材料相比, 其安全性较高。通过现代技术手段将SADs从水煎液中分离纯化, 然后进行理化表征和药理活性研究, 可为阐明中药“一碗汤剂”作用物质基础、药效物质吸收起效形式提供新角度。

2 中药水煎液自组装聚集体研究常用技术

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中药水煎液自组装聚集体是在热力学条件下, 活性成分由于复杂的相互作用自组装形成的超分子。目前对SADs的研究主要包括分离纯化、表征、吸收与药效等方面, 依据所研究水煎液不同相态特点和聚集体的性质, 选择恰当的方法对SADs进行分离纯化和表征, 研究SADs的形貌、粒径、电位、多分散系数等及其组成成分, 并利用核磁共振氢谱法、光谱法、X射线衍射法等技术对参与聚集体形成的分子间互作力进行分析, 研究其形成机制; 最后可通过细胞、外翻肠囊、单向肠灌流、整体动物等实验研究SADs的体内外吸收过程、药理活性及起效机制等。SADs研究常用技术见图 1。

2.1 分离纯化技术

中药水煎液中成分多样, 热诱导形成的聚集体尺寸、组成各异, 可根据目标聚集体的大小、电负性、稳定性、组成成分等选择合适的分离纯化方法。目前实验常用的超速/差速离心法根据聚集体重量、密度等性质的不同实现其分离和富集, 操作简单, 样品损失少; 蔗糖密度梯度离心法利用聚集体密度在惰性梯度介质中沉降速度不同分离。此外, 可采用尺寸排阻色谱法、透析法、超滤法等对不同大小的聚集体进一步分离纯化^[45]。根据目标聚集体所含蛋白质、多糖等含量差异, 可采用水提醇沉、丙酮沉淀等方法实现聚集体的快速分离, 但该法特异性低, 分离选择性差, 反复多次沉淀易造成聚集体损失, 醇/丙酮可能破坏粒子结构。具体可根据实验需要和目标聚集体的性质选择合适的分离纯化技术。

2.2 聚集体表征技术与自组装机制

目前研究主要通过透射电镜、扫描电镜等技术对聚集体的形貌进行观察, 动态光散射、纳米颗粒跟踪分析技术测定聚集体的粒径、电位等参数, 并结合高效液相或液质联用等技术对聚集体的组成成分进行定性、定量分析。此外, 由于蛋白、多糖成分广泛存在于中药水煎液中, 可采用蛋白质定量试剂盒法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳技术测定SADs中蛋白含量和分子量大小, 采用硫酸苯酚法等方法测定SADs中多糖含量。

Chen等^[46]采用液质联用法对黄连解毒汤上清液和自沉淀中的化学成分进行定性和定量分析发现, 自沉淀中小檗碱和黄芩苷含量显著高于上清液, 后续发现小檗碱和黄芩苷可自组装形成174 nm左右的纳米粒。此外, 陆续有研究报道小檗碱可和肉桂酸^[47]、大黄酸^[48]、马兜铃酸^[49]等有机酸自组装形成具有良好抗菌效果的纳米结构, 甘草与附子合煎过程甘草蛋白可通过与乌头碱自组装而达到减毒效果^[23]。Zhao等^[36]研究发现脑络心通汤自组装纳米粒由多糖、蛋白、人参皂苷Rg₁、人参皂苷Rb₁和黄芪皂苷IV等成分组成。在煎煮过程中, 中药成分相互作用的自组装行为与中医药配伍理论不谋而合, 以水为媒, 众药合和以为剂。

目前主要可通过紫外光谱法、傅里叶红外光谱法、荧光光谱法、核磁共振波谱法、X射线衍射法、圆二色光谱法和等温滴定量热法等方法推测SADs自组装机制。紫外吸收的变化可以证明共轭结构发生了变化, 红外光谱根据特征吸收峰可得到分子官能团信息, 通过分析紫外吸收和红外光谱的变化如峰的左右移动(红移/蓝移) 和峰的强弱(高度) 变化, 可得到分子自身结构、参与组装的关键官能团等分子间相互作用的信息^{[34, 50]}; 当存在荧光物质分子参与自组装时, 可利用荧光光谱技术研究分子相互作用后发生的荧光淬灭效应, 即荧光物质分子与其他分子相互作用引起荧光强度降低的现象。关于SADs荧光淬灭主要有两种形式: 动态淬灭和静态淬灭。静态淬灭指淬灭剂分子与荧光分子之间形成了新的复合物, 淬灭常数随温度升高而减小; 动态淬灭指淬灭剂分子与荧光分子的激发态分子之间相互碰撞而导致的荧光淬灭, 淬灭常数随温度的升高而增大, 遵循Stem-Volmer方程式(1):

(1)

$ F_{0}/F = 1 + K_\text{sv}[Q] = 1 + K_\text{q}τ_{0}[\text{Q}] $

式中, F₀和F分别为加入淬灭剂前后的荧光物质的荧光强度, K_sv为动态淬灭常数, [Q]为淬灭剂浓度, K_q为双分子淬灭常数, τ₀为淬灭体不存在时荧光分子平均寿命^{[34, 51]}。可通过检测在不同浓度下荧光物质分子荧光强度和吸收峰的变化以及不同温度下淬灭常数的变化, 分析参与SADs自组装的分子相互作用。

核磁共振波谱法检测到的氢原子/碳原子不同化学位移可以解释化学结构的变化; X射线衍射法可通过比较衍射图谱中特征峰的位置和相对强度变化, 推测分子空间结构的改变, 并以此分析可能发生的分子相互作用; 圆二色光谱法可用于进一步探索热力学对每个样品的旋光性的影响^{[52, 53]}。等温滴定量热法通过分析参与反应的物质的结合热和反应的热力学参数, 判断反应的自发性与分子相互作用。滴定后, 可使用纳米分析软件对数据进行分析, 在输入样品浓度后, 其自动搜索并拟合滴定曲线, 并计算出热力学参数。热力学参数由焓变(∆H)、熵变(∆S) 和结合常数(K_a) 组成, 吉布斯自由能变化(∆G) 采用标准热力学方程(2) 计算^{[34, 54, 55]}:

(2)

$ ∆G = -RT\ln K_\text{a} = ∆H - T∆S $

当∆G < 0时, 反应自发进行。通过分析∆H和T∆S之间的关系, 可推测驱动反应发生的主要作用力, 负焓和正熵有利于反应的自发进行。

Li等^[34]采用紫外光谱、傅里叶红外光谱和荧光光谱技术研究发现, 甘草蛋白与小檗碱通过静电引力和疏水作用等弱键连接形成, 并通过计算热力学参数发现, 当甘草蛋白与小檗碱作用时, ΔG < 0, ΔH > 0, ΔS > 0, 表明该过程为熵主导的自发吸热反应; Li等^[8]采用紫外光谱法、傅里叶红外光谱法、核磁共振波谱法和等温滴定量热法发现小檗碱-黄芩苷纳米颗粒的形成是由静电和疏水相互作用驱动的自发放热反应; Lin等^[56]采用等温滴定量热法研究发现大黄与黄连的相互作用是自发放热反应, 并采用扫描电镜、紫外光谱、傅里叶红外光谱、核磁共振波谱等技术发现大黄酸和黄连碱、大黄素和黄连碱可在静电吸引、氢键和π-π堆积诱导下分别自组装形成纳米纤维和纳米颗粒。

3 中药水煎液自组装聚集体研究相关问题

收起

上述研究中尚存在一些问题需要注意。首先, 中药水煎液体系中含有真溶液、胶体溶液、混悬液等不同分散体系, 具有多分散性。水煎液中的活性成分在加热过程中可发生多种物理相互作用和化学反应, 在一种或多种互作力和化学反应的驱动下, 活性成分可自组装形成大小不等、形貌各异的聚集体, 其质量均一性难以把控。其次, 对于自组装过程中参与的分子识别、互作力及相关化学反应等聚集体自组装规律亦有待揭示。再者, 从汤剂中分离得到的主要基于非共价作用力形成的聚集体, 往往存在物理化学稳定性较差的缺陷。聚集体在水煎液中形成、提取分离及放置过程中极易受到温度、加热时间、pH等因素的影响, 其不稳定性导致其临床应用受限。最后, 是产品分离纯化的纯度与产率的问题, 追求自组装纳米粒的高纯度虽然是科学研究的需要, 但并不利于产品的大规模生产, 甚至可能由于上述纯化方法的缺陷而导致药效学不理想或产率太低, 较之中药水煎液, 聚集体相对复杂的提取分离技术和高成本的制造需要被规范化评估, 详见图 2。

3.1 水煎液体系多分散性值得关注

中药多分散体系中存在分子、团簇、微/纳米聚集体等不同物质形态, 分离得到的不同相态粒度均一性难以保证。随着放置时间的延长, 聚集体可能会二次或多次聚集形成粒径更大的聚集物。此外, 由于可能存在的聚集、解聚、聚集体不稳定、部分化合物未参与自组装或自组装不完全等现象, 易导致不同批次聚集体质量均一性和稳定性存在差异, 研究者应对此多加关注。

中药煎煮过程中, 活性分子通过共价或非共价键等相互作用力自组装形成的聚集体大小不等, 形貌各异。利用现代技术手段如差速离心法对水煎液中聚集体进行分离, 难以保证目的产物均为理想尺寸的自组装颗粒。如Lin等^[42]采用超速离心法对葛根芩连汤自组装纳米粒进行分析, 发现汤中聚集体存在3种相态: 微聚体、微/纳米聚集体和上清液; Dou等^[39]采用膜透析和离心等方法, 在生脉饮中拆分得到了混悬相态、胶体溶液和全溶液3种不同相态, 并发现混悬相态粒径远大于其他相态。Wang等^[30]发现中药水煎液的浓度也会影响汤中形成聚集体大小, 在粉葛水煎液中, 聚集体粒径随水煎液浓度升高而增大, 且该研究发现, 聚集体粒径越小, 药物吸收越好。

除粒度外, SADs的形貌差异亦可对其生物利用度或药理活性产生直接影响。Shen等^{[37, 38]}发现芍药甘草汤中存在不规则球形纳米粒, 芍药单煎液中不存在纳米粒, 甘草单煎液和芍药甘草单煎合液中存在棒状或梭形粒子。与单煎液和单煎合液相比, 芍药甘草汤可显著改善甘草难溶性成分的体外释放, 促进甘草和芍药主要成分在回肠的吸收。有报道称球形纳米粒比棒状纳米粒更易吸收^[57]。Li等^[8]研究发现, 小檗碱和黄芩苷、汉黄芩苷相互作用, 可分别自组装形成纳米粒和纳米纤维, 并在抗菌活性方面表现出显著差异, 活性强弱为: 纳米粒 > 小檗碱 > 纳米纤维。

3.2 SADs自组装规律有待揭示

在中药煎煮过程中多种成分交织互作, 弱键诱导形成的自组装超分子体系是中药配伍的物质基础^{[34, 50]}, 由于较难进行分子间作用力的分析, 对于SADs自组装机制的认识大部分是基于其已知成分及其结构特点的合理推测^{[58, 59]}。研究SADs的形成机制, 即何种成分在何种分子互作力下形成了何种结构, 可为阐明中药复方配伍对汤剂中SADs形成的作用以及指导人工合成效优可控的SADs提供参考, 具体关于分子间作用力研究技术及原理详见2.2项。

对中药水煎液自沉淀现象和超分子自组装行为的研究发现^{[6, 60, 61]}, 生物碱类成分易和苷类、有机酸类、鞣质类等成分发生酸碱络合等相互作用而生成自沉淀; 鞣质类成分易和蛋白质类成分反应生成复合物; 蛋白和多糖的复杂长链结构受疏水作用、氢键作用等易发生分子折叠、翻转、螺旋等自组装行为; 皂苷兼具疏水基团(通常为三萜或甾体基团) 和糖亲水单元, 在临界胶束浓度条件下, 可聚集形成热力学稳定的“胶束”或“胶团”, 形成有序排列的聚集体。此外, 也有研究指出, 从能量的角度来看, 分子间的结合会导致能量减少, 这是结合的驱动力和趋势。为了减少系统的能量, 分子间结合通常是放热的^[54]。

当前SADs自组装规律有关理论研究尚不能满足实验研究的需要, 对于参与自组装形成SADs所需分子种类、配伍比例、成分与结构间关系、反应时间及温度等问题尚未形成易于把握的规律性认识, 这与此方面的实践研究缺少有关, 也与中药和复方水煎液成分的复杂多变有关, 亟须揭示SADs自组装机制及规律, 深入认识SADs分子自组装过程, 获悉自组装过程中参与的分子识别、互作力及相关化学反应, 甚至实现有限尺度地对自组装过程的调控, 从微观领域对中药学进行研究, 以促进中药新药开发和中药学现代化发展。

可以尝试由简入繁, 从成分已知、疗效显著的常用单味药入手, 从易发生酸碱络合等反应的临床常用药对入手, 从经典名方入手。也可在观察中药水煎液自沉淀的基础上, 通过分析自沉淀与上清液组成成分差异, 推测可能存在的自组装行为和参与其中的活性分子。如从调和诸药、能解百毒的甘草, “补气之圣药, 活人之灵苗”的人参, 擅清热燥湿、泻火解毒的黄连等代表药物入手, 研究其中所含鞣质类、蛋白多糖类、皂苷类等成分与有机酸类、萜类、生物碱类等成分可能产生的奇妙反应, 探究SADs自组装机制及规律。

3.3 影响SADs稳定性的因素有待研究

中药水煎液体系的多分散性和主要基于非共价作用力的自组装, 使得SADs往往存在物理化学稳定性较差的缺陷。此外, 制备、放置过程、加工温度、加热时间、pH等因素均会影响SADs的稳定, 在不同条件下聚集体稳定性变化不同。如仅在煮沸的蛤蜊汤中发现了聚集体, 而在蛤蜊的冷水提取物或冷水提取物的煮沸液体中均未发现, 这表明了加工温度是影响纳米粒形成的关键因素之一^{[28, 62]}。对于主要由蛋白等不耐热成分组成的聚集体, 可采用水浴加热回流或冷水浸提等方式提取聚集体, 以规避直接煎煮时高温可能对此类大部分聚集体产生的聚集、变性等风险。而Huang等^[53]研究发现热力学条件可影响小檗碱-黄芩苷自组装行为, 小檗碱和黄芩苷物理混合物会立即沉淀形成大尺寸的纳米纤维, 共煎后纳米纤维可转化和组装形成均一、稳定的纳米粒。

Zhou等^{[11, 14]}发现甘草汤和板蓝根水煎液中的自组装聚集体稳定性受到加热温度和pH的影响不同。加热温度越高, 甘草蛋白聚集体粒径越大, 而板蓝根蛋白聚集体平均粒径将减小; 甘草汤在pH 1.8、2.1、7.9和8.9下制备的聚集体平均粒径小于100 nm, 而在其他pH值下制备的样品粒径大于600 nm, 并有可见沉淀; 板蓝根水煎液仅在pH为5时, 聚集体平均粒径最小, 过酸(pH 2~3) 或过碱(pH 8~10) 都可能导致粒径增大2~3倍。Lin等^[13]发现较高pH、蛋白浓度和加热温度均有利于苦杏仁蛋白纳米粒的形成, 加热时间对纳米粒的形成影响较小。

中药复方汤液大多属于混合相态, 不同水煎液、不同制备方式所得水煎液、同一水煎液中不同相态存在差异, 不同活性成分可能存在于不同相态中, 在热力学和非共价相互作用的物理化学驱动下, 汤液中分子将不可避免地进行自身或与其他分子的自组装, 不同自组装形态会导致不同的生物活性^[5], 煎煮过程中自组装的不稳定性易导致所得聚集体质量不可控。对于中药水煎液来说, 药材产地、采收时间、炮制规范性、煎煮时长、火候等均会对水煎液的组成产生一定的影响。对于科学研究者来说, 需要固定饮片批次, 严格把控煎煮工艺、聚集体的提取分离条件, 一次性制备足够研究使用、稳定均一的自组装聚集体等, 全方位保证实验可重复性。

3.4 产品纯度与产率需要关注

随着对SADs分离纯化研究的深入, 产品纯度与产率问题越来越需要引起研究者的关注。目前常联用离心、超滤、尺寸排阻色谱、透析等多种方法, 分离纯化SADs, 以最大化除杂为目的, 获得尽可能均一纯净的自组装聚集体。其中差速离心法通过对不同尺寸和密度颗粒离心力的差异进行分离, 是分离纯化SADs的常用方法。不同离心条件分离得到的聚集体粒径大小和均一性不同, 如Fan等^[24]发现随着离心力的增加, 没食子汤剂中的聚集体平均粒径逐渐减小, 粒径更加均匀。Zeng等^[63]发现长离心时间是导致芦荟外泌体囊泡粒径不均匀的关键因素, 离心10和20 min分离得到的囊泡平均粒径相似, 约为200 nm左右, 多分散系数(polydispersity index, PDI) 分别为0.14和0.21, 随着离心时间的延长, 粒径和PDI均显著增加, 离心60 min后, 囊泡的最大粒径和PDI分别达到553.4 nm和0.59; 同时发现芦荟多糖导致的高黏度也会影响溶液中外泌体囊泡的分离效率, 研究人员通过优化离心时间与溶液黏度获得了高产量外泌体囊泡。

然而, 目前分离纯化SADs的方法均存在一些不足, 在研究时值得注意, 以根据实际情况选择合适的方法。如在高离心力下产生的壁效应可能会破坏SADs结构的完整性或导致聚集。可以采用向溶液介质中加入蔗糖或OptiPrep等物质, 通过不同密度梯度对离心过程进行缓冲, 以消除超速离心过程中的结构破坏和聚集, 保护SADs结构与形态。在超滤过程中, 聚集体易受剪切力的影响, 导致聚集体结块或团聚。与两种方法相比, 尺寸排阻色谱法不存在使聚集体二次聚集的风险, 且可通过监测不同时间段洗脱液粒径并收集合并粒径相同的洗脱液, 使得粒径大小均一性更为可控, 缺陷在于仅适用于少量样品自组装聚集体分离, 不适于大量制备。透析法是除去小分子化合物的常用方法, 可用来除去中药水煎液中未参与自组装的小分子化合物。然而, 透析液的选用、透析环境温度、透析时长等均会影响聚集体的稳定性与实验的重复性。透析时间较长, 会造成聚集体包载的活性成分在透析过程中释放出来, 导致聚集体成分含量偏低^[64]。此外, 透析环境基本为室温, 不同季节透析环境温度的差异性、透析存在的附着损失等问题均可能会对自组装聚集体的稳定性产生难以控制的影响。

追求自组装聚集体的高纯度虽然是科学研究的需要, 但并不利于产品的大规模生产, 甚至可能由于上述纯化方法的缺陷而导致药效学不理想或产率太低, 较之中药水煎液, SADs相对复杂的提取分离技术和高成本的生产也值得进一步思考。大规模、低成本、可重复的制备过程是满足生物医学研究和临床转化需求的重点, 如何把握好获得目的自组装聚集体纯度与产率之间的平衡, 建立合适的分离纯化方法, 值得研究与探讨。在掌握SADs自组装规律的基础上尝试对其进行人工合成可能是一条可行之路。

4 总结与展望

收起

汤剂, 具有随症加减治疗、起效迅速的优势, 是中医临床用药的主要形式。然而中药水煎液中成分众多, 其药效成分的存在形式与起效机制仍不明晰, 对于中药药效物质基础、复方配伍内涵仍有待进一步深入研究。SADs普遍存在于中药水煎液中, 并在药效发挥过程中起重要作用^[65]。较之单体成分, 煎煮时活性成分相互作用的自组装行为与中药配伍理论不谋而合, 且由多成分组成的SADs, 符合中医多靶点、整体性疗法的特点。对SADs的研究, 不再局限于单一的化学角度, 从成分互作-相态结构角度对汤液起效机制进行阐释, 为揭示中药水煎液复杂体系药效物质基础、作用机制与阐释传统方剂现代科学内涵提供了新思路。此外, 由于SADs是从中药水煎液中提取得到的, 无需复杂的化学修饰过程, 生物安全性与药理活性使其成为中药新药开发的全新策略。

目前, SADs的研究尚处于起步阶段, 其分离、纯化、理化性质、产品纯度与产率、质量控制、安全性和体内转运等有待综合考量和深入研究。可从物理药剂学以及新型给药系统、生物技术药物的研究中借鉴方法和手段, 以确定规范、标准化的制备方法, 建立合适的质量控制参数、储存方法等, 为阐明中药药效物质基础与提升中药新药研发水平提供有益的参考。

作者贡献: 王琪是本文的主要完成者; 郭小萌、李美景和徐睿协助查询相关文献并进行表格的整理; 倪乾坤、梁兴杰、龚慕辛指导论文的撰写并提出修改意见。

利益冲突: 所有作者均声明不存在利益冲突。

基金

收起

国家自然科学基金面上项目(81473360)
2020年北京市教委“实培计划”资助项目

参考文献

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2024年第59卷第1期

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doi: 10.16438/j.0513-4870.2023-0356

接收时间：2023-03-25
首发时间：2025-11-28
出版时间：2024-01-12

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出版历史

收稿日期：2023-03-25
修回日期：2023-06-01

基金

国家自然科学基金面上项目(81473360)

2020年北京市教委“实培计划”资助项目

作者信息

1.首都医科大学中医药学院, 北京 100069

2.中医络病研究北京市重点实验室, 北京 100069

3.国家纳米科学中心, 中国科学院纳米科学卓越创新中心, 中国科学院纳米生物效应与安全性重点实验室, 北京 100190

4.中国科学院大学, 北京 100086

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^*梁兴杰, E-mail: liangxj@nanoctr.cn;

龚慕辛, E-mail: gongmuxin@126.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Source	Isolation method	Size/nm	Function	Constituent	Ref.
Aqueous extract of Turkish gall	Differential centrifugation	Size decreased with the increasing centrifugal force, 6 577 (×g)-9 500 (×g) TG-LP NPs: 413.89 ± 202.95 TG-HP NPs: 230.34 ± 59.48	Potent pH responsiveness, free radical scavenging, and antibacterial effects	Galli cactechol oligomers	[24]
Black tea infusion	SEC	187 ± 7	Antioxidant activity	Protein, polysaccharides, caffeine, gallic acid and catechins	[25-27]
Freshwater clam (Corbicula fluminea Muller) soup	Centrifugation, SEC-DLS	Clam soup: 74.82 ± 0.68; fractions isolated by SEC F1: 70.86 ± 1.82 F2: 45.88 ± 4.16 F3: 50.35 ± 9.67	Hypolipidemic effect and improve nonalcoholic fatty liver disease	Taurine, ornithine and phytosterols; protein, carbohydrate and lipid	[10, 28]
Centrifugation, ultrafiltration	Clam soup: 77.52 ± 1.50
Aqueous extract of Armeniacae Semen Amarum	Centrifugation, dialysis, anion exchange and gel filtration chromatography, thermal induction	92	Encapsulate paclitaxel	Polypeptides	[13]
Coptidis Rhizoma decoction	Ultra-centrifugation and anion exchange chromatography	Fractions obtained by elution: CCD-P1: 272.7 ± 25.2 CCD-P2: 264.8 ± 21.4 CCD-P3: 427.4 ± 41.8	Promote intestinal berberine absorption	Mannose, rhamnose, glucose, galactose, arabinose and other sugars; galacturonic acid and little protein	[15]
Centrifugation, alcohol precipitation and Sevage method	Size increased with the increase of concentration (20-80 μg·mL^-1) CCP-P1: ~200 CCP-P2: ~380 CCP-P3: ~415	Promote intestinal berberine absorption; inhibit advanced glycation end product formation	[15, 29]
Aqueous extract of Glycyrrhizae Radix et Rhizoma	Precipitation with 50% ethanol-water solution, purified with anionic exchange chromatography followed by hydrophobic interaction chromatography, thermal induction	74.1 ± 0.7	Promote cellular uptake of Caco-2, L02, Hep-G2, macrophage NR8383 and proliferation of normal hepatocytes; solubilize the insoluble astragaloside IV by encapsulation	Protein	[11]
Isatidis Radix decoction	SEC-MALLS	~120	Promote the growth of normal cells but suppress that of cancerogenic cells and macrophages	Protein	[14]
Centrifugation, SEC-DLS	57-300	Antiviral effects	Polysaccharides	[16]
Puerariae thomsonii Radix decoction	Preparation of different concentrations of Fenge water decoction	Size increased with the increase of concentration (0.07-0.35 g·mL^-1) 0.07 g·mL^-1 (F-1): 120 0.10 g·mL^-1 (F-2): 195 0.17 g·mL^-1 (F-3): 256 0.35 g·mL^-1 (F-4): 480	Promote the absorption of ingredients in decoction and reduce AngⅡ level in acute blood stasis model rats	-	[30]
Aqueous extract of Rabdosia rubescentis leaves	Centrifugation	Size increased with the decrease of concentration (1.0-0.12 mg·mL^-1) 4.5-234.3	Inhibit thrombosis and antiplatelet aggregation	Nucleotides, diterpenoids, rosmarinic acid and other phenolic acids	[31]
Green tea infusion	Centrifugation, dialysis, SEC-HPLC	100-300	Immunostimulatory effect; load doxorubicin for drug delivery	Protein, polysaccharides	[32]
Puerariae lobatae Radix decoction	Ultra-centrifugation and filtration	200-351	Improve tiny circulation	Puerarin, daidzein, daidzin, genistein	[33]
Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma decoction	Water extract and acetone precipitation	185.5	Antibacterial activity	Protein, berberine	[34]
Glycyrrhizae Radix et Rhizoma and Aconiti lateralis Radix Praeparata decoction	Ethanol precipitation (40%-60%), anionic exchange chromatography and hydrophobic chromatography, adjust pH to 5 with HCl	Licorice root protein nanoparticles (GP NPs): 206.20 ± 2.02; GP-aconitine nanoparticles (GP-AC NPs): 238.20 ± 1.23	Reduce the toxicity of aconitine	Protein, aconitine	[35]
Naoluo Xintong decoction	Differential centrifugation and dialysis	200	Play an essential role in the brain-protective effect of decoction	Ginsenoside Rg₁, ginsenoside Rb₁, astragaloside IV, protein, polysaccharides	[36]
Shaoyao Gancao decoction	Ultra-centrifugation	200	Encapsulate the components of Baishao and facilitate the absorption of drugs in the ileum	Albiflorin, paeoniflorin and benzoylpaeoniflorin	[37, 38]
Shengmai Yin (pulse-activating decoction)	Centrifugation and dialysis	Decoction: 450.80 ± 113.61; colloidal phase: 87.53 ± 1.57; suspension phase: 2 938.67 ± 168.15	Alleviate the symptoms of heart failure in SD rats with deficiency of both qi and yin	-	[39]
Baihu decoction	Differential centrifugation and dialysis	100	Antipyretic effect	Neomangiferin, mangiferin, glycyrrhizic acid and ammonium glycyrrhizinate; C, O, Mg, Al, Si, Ca, Zn and other elements	[40, 41]
Ge-Gen-Qin-Lian decoction	Ultra-centrifugation	Microaggregates (MA): 2 775 ± 712; micro/nanoaggregates (MNA): 531 ± 23	Antioxidant cellular protection and improve the bioavailability of baicalin	-	[42]
Ma-Xing-Shi-Gan decoction	Centrifugation, SEC-MALLS	50-150	Influence cell viability	Ephedrine, pseudoephedrine	[43]
Centrifugation	S-13500: 170	Antibacterial activity	Ephedrine, amygdalin, glycyrrhizic acid and inorganic components Ca, K, Mg, etc	[44]

Source

Isolation method

Size/nm

Function

Constituent

Ref.

Aqueous extract of Turkish gall

Differential centrifugation

Size decreased with the increasing centrifugal force, 6 577 (×g)-9 500 (×g) TG-LP NPs: 413.89 ± 202.95 TG-HP NPs: 230.34 ± 59.48

Potent pH responsiveness, free radical scavenging, and antibacterial effects

Galli cactechol oligomers

[24]

Black tea infusion

SEC

187 ± 7

Antioxidant activity

Protein, polysaccharides, caffeine, gallic acid and catechins

[25-27]

Freshwater clam (Corbicula fluminea Muller) soup

Centrifugation, SEC-DLS

Clam soup: 74.82 ± 0.68; fractions isolated by SEC F1: 70.86 ± 1.82 F2: 45.88 ± 4.16 F3: 50.35 ± 9.67

Hypolipidemic effect and improve nonalcoholic fatty liver disease

Taurine, ornithine and phytosterols; protein, carbohydrate and lipid

[10, 28]

Centrifugation, ultrafiltration

Clam soup: 77.52 ± 1.50

Aqueous extract of Armeniacae Semen Amarum

Centrifugation, dialysis, anion exchange and gel filtration chromatography, thermal induction

Encapsulate paclitaxel

Polypeptides

[13]

Coptidis Rhizoma decoction

Ultra-centrifugation and anion exchange chromatography

Fractions obtained by elution: CCD-P1: 272.7 ± 25.2 CCD-P2: 264.8 ± 21.4 CCD-P3: 427.4 ± 41.8

Promote intestinal berberine absorption

Mannose, rhamnose, glucose, galactose, arabinose and other sugars; galacturonic acid and little protein

[15]

Centrifugation, alcohol precipitation and Sevage method

Size increased with the increase of concentration (20-80 μg·mL^-1) CCP-P1: ~200 CCP-P2: ~380 CCP-P3: ~415

Promote intestinal berberine absorption; inhibit advanced glycation end product formation

[15, 29]

Aqueous extract of Glycyrrhizae Radix et Rhizoma

Precipitation with 50% ethanol-water solution, purified with anionic exchange chromatography followed by hydrophobic interaction chromatography, thermal induction

74.1 ± 0.7

Promote cellular uptake of Caco-2, L02, Hep-G2, macrophage NR8383 and proliferation of normal hepatocytes; solubilize the insoluble astragaloside IV by encapsulation

Protein

[11]

Isatidis Radix decoction

SEC-MALLS

~120

Promote the growth of normal cells but suppress that of cancerogenic cells and macrophages

Protein

[14]

Centrifugation, SEC-DLS

57-300

Antiviral effects

Polysaccharides

[16]

Puerariae thomsonii Radix decoction

Preparation of different concentrations of Fenge water decoction

Size increased with the increase of concentration (0.07-0.35 g·mL^-1) 0.07 g·mL^-1 (F-1): 120 0.10 g·mL^-1 (F-2): 195 0.17 g·mL^-1 (F-3): 256 0.35 g·mL^-1 (F-4): 480

Promote the absorption of ingredients in decoction and reduce AngⅡ level in acute blood stasis model rats

[30]

Aqueous extract of Rabdosia rubescentis leaves

Centrifugation

Size increased with the decrease of concentration (1.0-0.12 mg·mL^-1) 4.5-234.3

Inhibit thrombosis and antiplatelet aggregation

Nucleotides, diterpenoids, rosmarinic acid and other phenolic acids

[31]

Green tea infusion

Centrifugation, dialysis, SEC-HPLC

100-300

Immunostimulatory effect; load doxorubicin for drug delivery

Protein, polysaccharides

[32]

Puerariae lobatae Radix decoction

Ultra-centrifugation and filtration

200-351

Improve tiny circulation

Puerarin, daidzein, daidzin, genistein

[33]

Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma decoction

Water extract and acetone precipitation

185.5

Antibacterial activity

Protein, berberine

[34]

Glycyrrhizae Radix et Rhizoma and Aconiti lateralis Radix Praeparata decoction

Ethanol precipitation (40%-60%), anionic exchange chromatography and hydrophobic chromatography, adjust pH to 5 with HCl

Licorice root protein nanoparticles (GP NPs): 206.20 ± 2.02; GP-aconitine nanoparticles (GP-AC NPs): 238.20 ± 1.23

Reduce the toxicity of aconitine

Protein, aconitine

[35]

Naoluo Xintong decoction

Differential centrifugation and dialysis

200

Play an essential role in the brain-protective effect of decoction

Ginsenoside Rg₁, ginsenoside Rb₁, astragaloside IV, protein, polysaccharides

[36]

Shaoyao Gancao decoction

Ultra-centrifugation

200

Encapsulate the components of Baishao and facilitate the absorption of drugs in the ileum

Albiflorin, paeoniflorin and benzoylpaeoniflorin

[37, 38]

Shengmai Yin (pulse-activating decoction)

Centrifugation and dialysis

Decoction: 450.80 ± 113.61; colloidal phase: 87.53 ± 1.57; suspension phase: 2 938.67 ± 168.15

Alleviate the symptoms of heart failure in SD rats with deficiency of both qi and yin

[39]

Baihu decoction

Differential centrifugation and dialysis

100

Antipyretic effect

Neomangiferin, mangiferin, glycyrrhizic acid and ammonium glycyrrhizinate; C, O, Mg, Al, Si, Ca, Zn and other elements

[40, 41]

Ge-Gen-Qin-Lian decoction

Ultra-centrifugation

Microaggregates (MA): 2 775 ± 712; micro/nanoaggregates (MNA): 531 ± 23

Antioxidant cellular protection and improve the bioavailability of baicalin

[42]

Ma-Xing-Shi-Gan decoction

Centrifugation, SEC-MALLS

50-150

Influence cell viability

Ephedrine, pseudoephedrine

[43]

Centrifugation

S-13500: 170

Antibacterial activity

Ephedrine, amygdalin, glycyrrhizic acid and inorganic components Ca, K, Mg, etc

[44]