中药药性理论是中医药理论的核心内容，是中药的性质、性能及其运用规律的高度概括，主要包含四气、五味、归经、升降浮沉、毒性等内容。中药药性理论是传统医药学中最有中国特色的理论，也是指导中医临床用药的核心理论依据。因此，应用现代科学技术深刻解读和诠释中药药性理论的科学内核，是推动现代中药学发展的重要基础与先导，将极大加速中药现代化进程，促进开发真正具有中医药特性和特色的现代化中药。

肠道菌群是指寄生在宿主肠道内、与宿主互利共生的微小生物体的集合体。大量研究表明，肠道菌群与人体证候有独特的对应关系，是人体证候的重要生物学指征，也是中药发挥其药效的关键媒介。首先，接受不同药性中药调节后的肠道菌群可以呈现出与药性相应的变化特征，随后，经过药性调节的肠道菌群进一步对人体各器官系统的功能进行调节，纠正机体的寒热证候偏倚，从而恢复人体的气机平衡并治疗疾病。肠道菌群能够体现并传递中药药性的潜在特征，为深入阐释中药药性的生物学内涵和体内传递机制提供了一个全新的思路和方向。此外，肠道菌群与中药的体内功效密切相关，一方面，肠道菌群可以分泌多种代谢酶，启动一系列重要的生物转化反应，从而对中药成分进行代谢，改变活性成分的循环强度以及中药的生物利用度；另一方面，某些中药可以调节肠道菌群的组成，改善菌群失调状态，进而恢复机体的正常生理机能，达到整体医治疾病的目的。因此，肠道菌群是介导多种中药及其活性成分发挥体内药理功效的重要媒介，通过考察药物与肠道菌群的相互作用，有助于深刻揭示中药，特别是口服生物利用度低的中药及活性成分发挥体内功效的作用机制，归纳不同药性中药诱导的肠道微生物组的特征可以预判中药新药药性。

本研究首先初步探索了肠道菌群变化特征与中药药性之间的关系，提出描述中药四性、五味、归经等药效特征与肠道菌群变化特征的对应关系。随后，以典型的味苦性寒中药黄连为例，探索了黄连及其活性成分小檗碱（BBR）对肠道微生物组的调节规律，为基于肠道微生物组的演变特征解析寒性药物作用机制提供了一个范例，主要研究结果如下：

第一部分：基于肠道菌群的中药药性理论解读

1. 肠道菌群对中药四性具有不同的响应

体内研究表明，不同药性的抗腹泻中药对肠道菌群有明显不同的影响，主要体现在：随着中药药性从热到寒，其对肠道菌群的影响逐渐增强，其中热性中药对肠道菌群的影响微弱，而寒性中药显著改变了肠道菌群的结构。在菌群组成方面，促炎菌Anaerotruncus，Tyzzerella和Ruminiclostridium在热性药物组富集，而抗炎菌Ruminococcaceae_UCG-010，Parasutterella和Bifidobacterium在寒性药物组富集，这与寒凉中药大多可以清热解毒，而热性药物常会导致上火的临床表现相一致。以上结果提示中药的寒热药性与肠道菌群关系密切，寒性中药的药理功效可能部分是通过调节肠道菌群实现的，而热性中药的体内功效可能更多地是通过其他途径实现。

2. 肠道菌群共丰度基因集（CAGs）对中药药味具有不同的响应

化学成分分析表明，黄酮、生物碱和皂苷分别是甘味、苦味和辛味中药中最丰富的成分，而复合药味的药物（苦-辛味和甘-辛味）表现出混合的成分。肠道菌群分析发现，同一药味中药对肠道菌群分类学的调节没有特定的规律，而CAGs对不同药味具有不同的响应，并且，这种差异性响应与它们的药理作用相关。提示CAG或可作为表征中药药味的潜在指标。

3. 中药以归经依赖性的方式逆转黄连对肠道菌群的改变

中药讲究配伍，以实现增效减毒的目的。体内研究表明，口服黄连会显著破坏小鼠肠道菌群，降低菌群的多样性。使用不同的中药与黄连进行配伍联合，可以在不同程度上减轻黄连对肠道菌群的不良影响。有趣的是，不同中药改善黄连对肠道菌群破坏的能力与其归经存在密切的关联，具体表现为：少阴经（黄柏、细辛、肉桂）中药与黄连联用使肠道菌群仅有轻度恢复，太阴经中药（升麻、白芷、石膏）和厥阴经中药（柴胡、独活、羌活）对肠道菌群的恢复能力更强，尤其是厥阴经中药使肠道菌群基本恢复到正常状态。黄连使Firmicutes（Clostridia和Bacilli）和Actinobacteria（Bifidobacteriales）降低，而使 Proteobacteria（Enterobacteriaceae）升高。少阴、太阴、厥阴经的中药与黄连联用可以逆转这种变化，使其逐渐恢复正常水平。此外，少阴、太阴、厥阴经的中药与黄连联用还保留了黄连对有益菌Akkermansia muciniphila和Blautia coccoides的富集作用。

4. 肠道真菌对清热药和解表药具有不同的响应

清热与解表中药虽均有抗菌、退热的功效，但在具体的临床应用上又有明显不同。为了揭示清热药与解表药的作用效果差异，本研究考察了这两类药对肠道细菌和肠道真菌的影响。小鼠体内研究表明，口服黄连显著改变了肠道细菌的结构，但对肠道真菌的影响不大。清热药或解表药与黄连联合使用以相似的方式减轻了黄连对肠道细菌的影响；同时，清热药或解表药与黄连联合使用均显著增强了黄连对肠道真菌的影响，并且后者优于前者。黄连与清热药联合使用富集真菌Penicillium，而与解表药联合使用富集真菌Fusarium。此外，解表药富集的Fusarium与细菌Akkermansia显著正相关。Akkermansia是一种与toll样受体4（Toll-like receptor 4，TLR4）相互作用，调节产热的有益菌。以上结果为解表药提供了肠道微生物相关的药理机制研究思路。

第二部分：基于肠道菌群的苦寒中药黄连及其活性成分小檗碱的降脂机制研究

5. 苦寒中药黄连对肠道细菌和真菌的双重调节作用与其抗高脂血症作用相关

药理研究结果显示，黄连显著降低高脂饮食（HFD）喂养小鼠的血脂水平，抑制其脂质积累。肠道微生物组检测发现，黄连降低了肠道细菌和真菌的多样性，同时改变了它们的组成。具体而言，黄连显著降低了Aspergillus spp.（A. chevalieri，A. luteovirescens，A. oryzae，A. sp. F51）和Penicillium spp.（P. expansum，P. janthinellum，P. sp. BAB-5649和P. sp. GZU-BCECYN66-5），而增加了 Tilletia bornmuelleri和Tilletia bromi。此外，肠道真菌和细菌之间还存在着复杂的联系。例如，真菌Aspergillus（A. chevalieri，A. luteovirescens，A. oryzae）与细菌Blautia coccoides负相关，而与Lactobacillus（L. johnsonii，L. sparagasseri，L. taiwanensis，L. amylovorus）正相关。这些结果表明，黄连可能通过改变肠道细菌和真菌组成，以及调节其相互作用来发挥抗高脂血症作用。

6. 肠道菌群特异性介导黄连活性成分小檗碱的抗高脂血症作用

基于改变给药方式，抗生素处理以及粪菌移植实验等手段，本研究证明肠道菌群是BBR发挥降脂功效的充分必要条件，其中，短链脂肪酸（SCFAs）产生菌，如Clostridium XI，Anaerostipes，Blautia，Akkermansia和Coprobacillus的增加，与BBR改善脂质代谢的功效紧密相关。此外，BBR还降低了Alistipes，Helicobacter，Enterorhabdus 和Desulfovibrio的相对丰度。以上结果表明肠道菌群可以特异性地介导BBR的抗高脂血症作用。

7. 小檗碱富集的Blautia producta改善了高脂血症，并上调了肝脏LDLR表达

与前述研究结果一致，口服BBR（200 mg/kg）显著降低了HFD诱导小鼠的血脂和肝脂水平。肠道菌群测序分析结果显示，Blautia与BBR的降脂功效密切相关。进一步分析发现，BBR可以选择性的促进SCFA产生菌Clostridium XIVa和Blautia producta在小鼠肠道的增殖，增加SCFAs的水平。将Blautia producta灌胃给予高脂饮食诱导小鼠，发现该菌种可以显著减轻小鼠肥胖，改善其高脂血症症状。值得注意的是，Blautia producta还上调了小鼠肝脏低密度脂蛋白受体（LDLR）的表达。基于HepG2细胞的体外实验同样发现，Blautia producta培养上清液可以上调LDLR水平，促进HepG2细胞对LDLR的摄取。该研究不仅鉴定了介导BBR降脂功效的具体菌种，还将BBR对肠道菌群的调节与对肝脏LDLR表达的上调联系起来，为BBR抗高脂血症的机制提供了新的见解。

8. 小檗碱通过刺激肠道粘蛋白分泌间接促进Akkermansia的生长

本研究发现BBR以剂量依赖性的方式增加了小鼠肠道Akkermansia的丰度。当体外直接孵育时，这种促生长作用被消除，表明BBR可能以宿主依赖的方式促进Akkermansia生长。进一步的研究发现BBR可显著增加结肠粘蛋白产生基因的转录和粘蛋白分泌。非靶向代谢组学分析表明，BBR增加粪便多胺水平，已知多胺可以刺激杯状细胞增殖和分化。但多胺合成酶抑制剂DFMO处理并没有消除BBR对粘蛋白分泌和Akkermansia生长的促进作用，表明肠道细菌来源的而非宿主来源的多胺可能参与BBR对Akkermansia的促生长作用。

综上所述，本研究首次探索了肠道菌群与中药药性理论的内在关联，为中药药性理论的现代化阐释指明了一个新方向。通过以苦寒中药黄连及其活性成分BBR为例，发现了寒性中药通过调节肠道微生物组，从而发挥抗高脂血症作用的分子机制，证实了基于肠道菌群变化特征预测药性的新方法具有足够的可行性。该研究结果将促进对中药传统理论的现代化理解，为中药药理机制的阐释提供新的研究方向。

The property theory is a basic theory of traditional Chinese medicine (TCM) that generalize the properties, actions and application rules of TCMs, including four qi, five flavors, meridian, ascending and descending, floating and sinking, and toxicity. The property theory is not only the most characteristic theory of TCM, but also a core theoretical basis for guiding the clinical use of TCM. The interpretation of property theory by modern science and technology is an important basis and forerunner of the modern development of TCM, and can guide the research of all aspects of TCMs.

Gut microbiota is a collection of microorganisms that inhabit host intestine. Numerous studies have demonstrated that the gut microbiota is an important biological indicator for human body syndrome, which can be used as the key medium for the transmission of TCM’s property in the body. On the one hand, the gut microbiota modulated by TCMs with different property showing the corresponding characteristics. On the other hand, the gut microbiota altered by TCMs further regulates the function of the human body, corrects the cold and hot changes of the body, so as to restore the balance of Qi movement to treat diseases. The gut microbiota reflects and transmits the properties of TCM, which provides a new idea and direction for the deep interpretation of the biological connotation and in vivo transmission mechanism of TCMs. Simultaneously, gut microbiota is also closely related to the efficacy of drugs. The gut microbes can metabolize herbal constituents into molecules with increased or decreased bioavailability and efficacy, thus alter the circulating intensity of the herb’s active components. On the other aspect, herbal medicines may change the composition of the gut microbiota, thereby improve the global functions of our body and remotely ameliorate diseases by enhancing beneficial germs and/or inhibiting bad germs.

This study preliminarily explored the relationship between the TCM property and gut microbiota, thus proposed the corresponding relationship between the characteristics of four natures, five flavors, meridian and the alteration of gut microbiota. Then, we took bitter-flavored and cold-natured TCM Coptis chinensis Franch. as an example, and explored the regulation pattern of Coptis chinensis Franch. and its active component berberine (BBR) on gut microbiome, which provided a paradigm for exploring the mechanism of cold-natured drugs based on the alteration of gut microbiome. The dominant outcomes of present study are as follows:

Section I: Interpreting the property of traditional Chinese medicine by gut microbiota-drug interaction

1. Traditional Chinese medicines differentially modulate the gut microbiota based on their nature (Yao-Xing)

The in vivo study showed that antidiarrheal TCMs with different natures showed distinct impacts on the gut microbiota. Specifically, the alteration of gut microbial structure was gradually stronger with the herbal nature turned from hot to cold, among which hot-natured TCMs had no influence on the gut microbiota but cold-natured TCMs notably changed the structure of the gut microbial community. As for composition, pro-inflammatory bacteria Anaerotruncus, Tyzzerella and Ruminiclostridium were enriched by hot-natured TCMs, while that of anti-inflammatory bacteria Ruminococcaceae_UCG-010, Parasutterella and Bifidobacterium were increased by cold-natured TCMs, which consistent with their efficacy. The above results suggested that cold TCMs may exert their role by modulating the gut microbiota, while hot TCMs may through other pathways.

2. The gut microbial co-abundance gene groups (CAGs) differentially respond to the flavor (Yao-Wei) of traditional Chinese medicine

The chemical component analysis showed that flavones, alkaloids, and saponins were the richest components for sweet-, bitter-, and pungent-flavored TCMs, respectively. Medicines with merged flavors (bitter-pungent and sweet-pungent) displayed mixed profiles of components. According to gut microbial analysis, modulation of TCMs belonging to the same flavor on the taxonomic classification was inconsistent to an extent, while the functional sets of gut microbiota, co-abundance gene groups (CAGs), strongly and differentially responded to distinct flavors. Moreover, these correlations were in line with their pharmacological actions. This indicated that the functional sets, CAGs, could be an appropriate indicator for TCM flavors.

3. TCMs with varied meridians (Gui-Jing) differentially alleviate the adverse impact of Coptis chinensis on gut microbiota

Coptis chinensis administration dramatically disrupted the gut microbiota and decreased the alpha diversity. Co-administration of different TCMs alleviated the adverse impact of Coptis chinensis on gut microbiota in a meridian-dependent manner. TCMs belonging to Shaoyin meridian moderately shifted the gut microbiota, while TCMs belonging to Taiyin and especially Jueyin meridians remarkably recovered the gut microbial community to the normal status. Decreased Firmicutes (Clostridia and Bacilli) and Actinobacteria (Bifidobacteriales) and increased Proteobacteria (Enterobacteriaceae) were main features of Coptis chinensis-induced dysbiosis. TCMs belonging to Shaoyin, Taiyin and Jueyin meridians gradually reversed the changes of these bacteria to the normal condition. Simultaneously, the enriching effect of Coptis chinensis on functionally beneficial bacteria such as Akkermansia muciniphila and Blautia coccoides was preserved when co-administration of Shaoyin, Taiyin and Jueyin meridians TCMs.

4. Gut fungi differentially response to the antipyretic (heat-clearing) and diaphoretic (exterior-releasing) TCMs in Coptis chinensis-conditioned gut microbiota

Oral administration of Coptis chinensis significantly changed the structure of gut bacteria but showed little influence on gut fungi. Co-treatment with antipyretic or diaphoretic TCMs alleviated the impact of Coptis chinensis on gut bacteria to a similar degree. However, combined with either antipyretic or diaphoretic TCMs significantly strengthened the influence of Coptis chinensis on gut fungi, with the latter superior to the former. The antipyretic TCMs enriched Penicillium spp. while diaphoretic TCMs promoted Fusarium spp. Further analysis revealed that the diaphoretic TCMs-enriched fungi Fusarium spp. were positively related to Akkermansia spp., a beneficial bacteria that interacts with Toll-like receptor 4 (TLR4) and regulates thermogenesis, thus providing a potential bacteria-related mechanism of diaphoretic TCMs.

Section II: Elucidating the lipid-lowering mechanism of berberine by modulating gut microbiota

5. Dual modulation of gut bacteria and fungi manifests the gut-based anti-hyperlipidemic effect of Coptis chinensis

Pharmacological results showed that Coptis chinensis significantly decreased serum lipids and inhibited the hepatic lipid accumulation in the HFD-fed mice. Mechanistically, Coptis chinensis reduced the diversity of gut bacteria and fungi, as well as changed their composition. Fungus Aspergillus species (A. chevalieri, A. luteovirescens, A. oryzae, A. sp. F51) and Penicillium (P. expansum, P. janthinellum, P. sp. BAB-5649 and P. sp. GZU-BCECYN66-5) were decreased in Coptis chinensis-treated group, while Tilletia bornmuelleri, Tilletia bromi were increased. Furthermore, there are complex association between intestinal fungi and bacteria. For example, fungus Aspergillus (Aspergillus chevalieri, Aspergillus luteovirescens, Aspergillus oryzae) was negative associated with bacterium Blautia coccoides, but positive associated with Lactobacillus (L. johnsonii, L. sparagasseri, L. taiwanensis, L. amylovorus). These results suggested that Coptis chinensis may exert anti-hyperlipidemic effects by altering gut bacterial and fungal composition as well as modulating their interactions.

6. Gut microbiota specifically mediates the anti-hyperlipidemic effect of BBR

Through different administration approach, antibiotics treatment and fecal microbiota transplantation, we found gut microbiota is necessary and sufficient for BBR’s hyperlipidemia-ameliorating effect. The enhancement of short chain fatty acids (SCFAs)-producing bacteria such as Clostridium XI, Anaerostipes, Blautia, Akkermansia and Coprobacillus is tightly associated with its anti-hyperlipidemia effect. Furthermore, BBR also decreased the relative abundance of Alistipes, Helicobacter, Enterorhabdus and Desulfovibrio. The above results indicate that the gut microbiota can specifically mediate the anti-hyperlipidemic effects of BBR.

7. The BBR-enriched gut commensal Blautia producta ameliorates high-fat diet (HFD)-induced hyperlipidemia and stimulates liver LDLR expression

Consistent with previous study, orally administration of BBR (200 mg/kg) decreased serum and liver lipid levels in HFD-induced hyperlipidemic mice. Microbiome analysis indicated that Blautia was closely associated with BBR’s lipid-modulating activities. Further analysis revealed that BBR selectively promoted the growth of SCFA-producing bacteria Clostridium XIVa and Blautia producta. Orally treatment of HFD mice with live Blautia producta reduced obesity and alleviated hyperlipidemia. Notably, Blautia producta significantly increased LDLR expression in the liver, and its culture supernatant upregulated the LDLR level and promoted LDL uptake by HepG2 cells. This study not only identified the specific strains that mediate the lipid-lowering efficacy of BBR, but also linked the modulation effect of BBR on gut microbiota and the upregulation of hepatic LDLR expression, thus providing new insights into the anti-hyperlipidemic mechanism of BBR.

8. Berberine, a potential prebiotic to indirectly promote Akkermansia growth through stimulating gut mucin secretion

Gavage of BBR dose-dependently increased the abundance of Akkermansia in mice. However, it did not stimulate Akkermansia growth in direct incubation, suggesting that BBR may promote Akkermansia in a host-dependent way. Oral administration of BBR significantly increased the transcription of mucin-producing genes and mucin secretion in colon. Untargeted metabolomics analysis showed that BBR increased polyamines production in feces which are known to stimulate goblet cell proliferation and differentiation, but treatment with eukaryotic polyamine synthase inhibitor DFMO did not abolish the stimulating effect of BBR on mucin secretion and Akkermansia growth, indicating that the gut bacteria-derived but not the host-derived polyamines may involve in the BBR-promoted Akkermansia growth.

In conclusion, this study explored the inherent association between the gut microbiota and the property theory of TCM for the first time, which laid a foundation for the modern interpretation of the property theory of TCM. Meanwhile, taking Coptis chinensis and its active component BBR as an example, we explored the potential molecular mechanism of their anti-hyperlipidemia effect based on the gut microbiome. This work will promote the understanding of TCM’s classic theory and provide new research directions for the interpretation of the pharmacological mechanisms of some TCMs.