Multidimensional Research on Bovine Bile Acid: From Traditional Medicinal Value to Modern Pharmacological Mechanisms

Bovine Calculus Bovis, a precious traditional Chinese medicine, has been widely used since ancient times for its therapeutic effects in clearing heat and detoxifying, opening orifices and resolving phlegm, and calming wind and spasms. Its core active component, "Bovine Calculus Bovis Bile Acids" (Bovine Bile Acids), is a complex mixture of bile acids with pharmacological effects that encompass anti-inflammatory, immunomodulatory, hepatoprotective, choleretic, and central nervous system regulatory functions. However, due to its complex chemical composition, natural origin, and significant quality variability, the pharmacological mechanisms and clinical applications of Bovine Bile Acids still require in-depth investigation. 

With the advancement of modern pharmacology, molecular biology, and analytical techniques, research on Bovine Bile Acids has gradually shifted from traditional empirical approaches to a more scientific and systematic direction. This review aims to systematically summarize the chemical composition, pharmacological actions, extraction and analytical methods, clinical applications, and future research directions of Bovine Bile Acids, to provide theoretical support and practical guidance for further research and application in this field. 

Definition, Chemical Basis, and Major Components of Bovine Bile Acids Modern chemical and pharmacological research has established that "Bovine Bile Acids" does not refer to a single chemical entity, but rather is a general term for a class of bioactive bile acid compounds in the traditional and precious herb "Bovine Calculus Bovis" (Bovine Calculus Bovis). Bovine Calculus Bovis, as a gallstone in the gallbladder of cattle, has a long history of medicinal use [1,2]. Therefore, the "Bovine Bile Acids" discussed in this review are essentially bioactive complexes primarily composed of various bile acids and their derivatives derived from Bovine Calculus Bovis or bovine bile [1,3-5]. The establishment of this understanding has gone through a process of scientific development: early studies once mistakenly judged that the main component of Bovine Calculus Bovis was inorganic salts, while subsequent in-depth chemical analysis fundamentally corrected this view and established bile acid substances as its main pharmacological basis [1,4]. 

Major Chemical Composition of Bovine Bile Acids 

The chemical composition of Bovine Calculus Bovis is highly complex, yet the core contributors to its pharmacological activity primarily fall into two major categories: bile acid compounds and bilirubin compounds, with bile acid compounds constituting the main body of "Bovine Bile Acids" [1-3]. Quantitative analysis indicates that the total content of bile acids in Bovine Calculus Bovis accounts for approximately 5.57% to 10.66% of its dry weight [5]. These components form a complex mixture, encompassing primary bile acids directly synthesized in the liver (such as cholic acid and chenodeoxycholic acid) and secondary bile acids transformed by the action of intestinal microbiota (such as deoxycholic acid, with a content of about 1.96% to 2.29%) [3,4], and are often present in the form of conjugated bile acids combined with glycine or taurine, such as glycocholic acid and taurocholic acid [4,5]. The establishment of this chemical framework can be traced back to the pioneering research on Bovine Calculus Bovis by the 19th-century chemist Adolf Strecker, whose successful isolation of bile acids laid the scientific foundation for modern understanding [6,7]. Meanwhile, bilirubin compounds, especially bilirubin with a content as high as 10% to 50%, represent another crucial active component in Bovine Calculus Bovis. Bilirubin and its oxidation products not only impart the characteristic color to Bovine Calculus Bovis but also possess significant antioxidant and anti-inflammatory activities, often exerting synergistic effects with bile acid components in pharmacological actions [1,2]. In addition, Bovine Calculus Bovis also contains cholesterol, various amino acids, peptides, and trace elements, which together constitute the complex and exquisite pharmacological material basis of Bovine Calculus Bovis. 

Molecular Structure and Physicochemical Properties of Core Bile Acids 

The various bile acid molecules that constitute "Bovine Bile Acids" share a fundamental structural framework, which directly determines their unique physicochemical properties and biological functions [8,9]. All bile acids are steroid compounds, with a rigid "perhydrocyclopentanophenanthrene" steroid nucleus formed by the fusion of four rings at their core. A flexible carboxylic acid side chain is attached to the C17 position of ring D [8], and the carboxyl group at the end of this side chain typically dissociates under physiological pH conditions, endowing the molecule with acidic and hydrophilic characteristics. Crucially, the substitution pattern of hydroxyl groups on the steroid nucleus—specifically, the number and spatial configuration (α or β) of hydroxyl groups at positions such as C3, C7, and C12—is the key factor distinguishing different bile acids and precisely regulating their hydrophobic-hydrophilic balance. For instance, cholic acid (molecular formula C₂₄H₄₀O₅) has α-configured hydroxyl groups at positions C3, C7, and C12, which gives it a relatively high hydrophilicity; in contrast, deoxycholic acid, lacking a hydroxyl group at position C7, exhibits greater hydrophobicity [9]. This unique structure—a rigid hydrophobic steroid core combined with polar hydrophilic hydroxyl/carboxyl groups—endows bile acid molecules with significant amphipathicity. This property allows them to spontaneously aggregate at the oil-water interface, functioning as an efficient natural biological surfactant to emulsify dietary fats in the intestine, which is one of their most fundamental physiological functions [10,11]. Moreover, in the body, the vast majority of bile acids are conjugated with glycine or taurine via an amide bond. This conjugation process significantly enhances the molecule's water solubility and greatly reduces its acid dissociation constant (pKa), ensuring that they remain ionized in the neutral to weakly alkaline environment of the intestine, thereby enabling them to perform their physiological functions more stably and effectively [11,12].

Traditional Chinese medicine theory holds that Bovine Calculus Bovis possesses the effects of clearing heat and detoxifying, calming wind and spasms, and opening orifices and resolving phlegm. Echoing this, animal bile (such as bovine and porcine bile) also has a long history of medicinal use in China and other traditional medical systems, often employed to treat eye diseases, inflammation, and digestive system disorders. Modern research, however, has focused on its main active components—bile acids—using scientific methods to verify and elucidate these traditional effects. Studies have shown that the pharmacological actions of Bovine Bile Acids are the result of the synergistic effects of their multi-component, multi-target characteristics. 

Anti-inflammatory, Antioxidant, and Immunomodulatory Functions 

Among the various pharmacological activities of Bovine Bile Acids, anti-inflammatory, antioxidant, and immunomodulatory effects have been the most actively studied in recent years. Taking glycocholic acid, an important component of Bovine Bile Acids, as an example, it has been proven to have significant anti-inflammatory, antioxidant, and immunomodulatory functions. Its mechanisms of action involve multiple levels: First, bile acids can inhibit the activation of key inflammatory signaling pathways such as nuclear factor-κB (NF-κB), reducing the production of downstream pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby exerting a direct anti-inflammatory effect [13]. Second, as a natural ligand of the farnesoid X receptor (FXR), bile acids can induce the expression of a series of target genes involved in bile acid metabolism, lipid metabolism, and negative regulation of inflammation after activating FXR, thus playing a systemic role in suppressing inflammation and protecting tissues [14-16]. In addition, Bovine Bile Acids also show clear antioxidant stress capabilities, effectively scavenging free radicals and enhancing the activity of endogenous antioxidant enzymes such as superoxide dismutase (SOD), thereby reducing oxidative stress damage to cells [16]. These series of molecular events are highly consistent with the traditional effect of Bovine Calculus Bovis in "clearing heat and detoxifying" at the biological level. 

Hepatoprotective and Choleretic Effects 

Hepatoprotective and choleretic effects are the most classic and fundamental pharmacological activities of Bovine Bile Acids. As the main organic component of bile, its mechanisms in this regard are mainly reflected in three aspects: First, it directly promotes bile secretion and excretion. Bile acids themselves can effectively stimulate hepatocytes to secrete bile, increasing bile flow and producing a clear "choleretic" effect, which helps to flush the biliary tract and promote the excretion of metabolic waste and toxins [17-19]. Second, it protects hepatocytes. Certain specific bile acids, such as ursodeoxycholic acid, can replace endogenous bile acids with strong hydrophobicity and higher cytotoxicity, thereby reducing the overall cytotoxicity of bile, stabilizing the structure of hepatocyte membranes, and exerting a direct protective effect [19,20]. Third, it regulates cholesterol metabolism. As the main terminal pathway for cholesterol catabolism in the body, Bovine Bile Acids play a core role in maintaining cholesterol homeostasis by regulating the rate of conversion of cholesterol to bile acids, which is of great significance in preventing the formation of cholesterol stones [21,22]. 

Effects on the Central Nervous System 

The traditional effects of Bovine Calculus Bovis in "calming wind and spasms, and opening orifices and resolving phlegm" clearly point to its potential impact on the central nervous system. Although the exact molecular mechanisms still need to be further explored, reasonable inferences can be made based on the characteristics of its components. Some bile acids, due to their relatively low molecular weight and certain amphipathic properties, theoretically have the potential to cross the blood-brain barrier. They may exert sedative, anticonvulsant, and other neuroregulatory effects indirectly or directly by regulating the Brain's γ-Aminobutyric Acid (GABA) and other neurotransmitter systems, affecting the excitability of neuronal cell membranes, or through reducing neuroinflammation [23]. Uncovering its exact targets and pathways of action in the central nervous system is a highly valuable research direction for the future [24,25]. 

Other Potential Pharmacological Activities 

In addition to the core activities mentioned above, Bovine Bile Acids also exhibit other potential pharmacological activities. Their most basic physiological function is reflected in the regulation of the digestive system. As an efficient biological surfactant, it emulsifies dietary fats in the intestine, which is a key substance for promoting the absorption of lipids and fat-soluble vitamins [26,27]. Moreover, with its unique amphipathic properties and good biocompatibility, bile acids and their derivatives are being widely explored as drug delivery carriers to construct novel drug delivery systems, in order to improve the solubility and bioavailability of poorly soluble drugs, demonstrating its broad application prospects in modern pharmaceutical science [28,29].

Transforming Bovine Bile Acids from complex natural products into standardized materials suitable for modern research and drug development relies heavily on efficient extraction and purification processes, precise analytical techniques, and a stringent quality control system. 

Extraction and Purification Processes 

Extraction and purification are the primary steps in obtaining high-purity bile acid mixtures or individual compounds [30]. Modern processes typically involve several stages [31]. Initially, the raw bovine bile material is pre-treated to remove large-molecule impurities through operations such as deproteinization and centrifugation [32]. Subsequently, adsorbents like activated carbon are utilized for decolorization and impurity removal to eliminate interferences from pigments such as bilirubin [33]. Next, precipitation methods such as salting out or acid precipitation are employed to separate bile acids from the solution, achieving preliminary separation from water-soluble impurities [30]. In the fine purification stage, traditional multiple recrystallization methods are increasingly being supplemented or replaced by more efficient column chromatography techniques (e.g., silica gel chromatography), which can achieve finer separations based on the differences in polarity among various bile acids, thereby obtaining total bile acids or high-purity individual bile acid monomers (such as cholic acid) [34]. 

Modern Analytical Techniques 

Accurate qualitative and quantitative analysis of the complex system of Bovine Bile Acids is crucial for ensuring the quality and reproducibility of their pharmacological effects [35]. Modern analytical techniques play a central role in this regard. Among them, chromatography-Mass Spectrometry (MS) has become a powerful tool for analyzing the bile acid profile [36]. For instance, ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS) leverages the high-efficiency separation capability of liquid chromatography to separate the various components in complex mixtures and then uses high-resolution MS to precisely determine their molecular weights and conduct structural identification, enabling rapid qualitative analysis of dozens of bile acids in bovine bile [37]. Additionally, spectroscopic methods such as Infrared Spectroscopy (IR) are often employed to assist in the analysis of functional group characteristics and structural identification of bile acid mixtures [38]. 

Challenges in Quality Control 

However, the quality control of Bovine Bile Acids faces significant challenges, primarily due to their natural source—Bovine Calculus Bovis [4]. As a biogenic calculus, its chemical composition and proportions are significantly influenced by various factors, including the breed, age, health status, origin, and calculus formation time of the cattle, leading to substantial batch-to-batch variations [39,40]. Therefore, future quality standards must go beyond merely specifying the content of total bile acids and establish a comprehensive quality control system based on "fingerprinting" or multi-component simultaneous quantification techniques to precisely monitor key active components such as cholic acid, deoxycholic acid, glycocholic acid, and bilirubin, ensuring the uniformity and reliability of different batches in terms of chemical composition and clinical efficacy [33,41].

The clinical application of Bovine Bile Acids has a long history, but its positioning in the modern medical system, safety, and corresponding regulatory policies constitute the practical framework for transforming traditional wisdom into safe and effective drugs [42]. 

Currently, the main clinical application of Bovine Bile Acids is reflected in the form of Bovine Calculus Bovis or artificial Bovine Calculus Bovis, as a core component in many Traditional Chinese Medicine (TCM) compound formulations. For example, well-known formulations such as Angong Niuhuang Pill and Niuhuang Qingxin Pill utilize their pharmacological activities of clearing heat, calming, and anti-inflammatory effects to play a unique role in treating severe conditions such as high fever, convulsions, and stroke [43,44]. In addition, based on a deeper understanding of the physiological functions of bile acids, some high-purity bile acid monomers (such as ursodeoxycholic acid) have also been officially used as Western medicines to treat cholestatic liver disease and cholesterol stones [17]. 

Despite a long history of medicinal use, systematic and modern toxicological data on Bovine Bile Acids mixtures are still relatively limited, and their safety cannot be ignored. The primary issue lies in the complexity of their components, as different bile acids have varying physiological activities and potential toxicities. For example, hydrophobic bile acids such as deoxycholic acid and lithocholic acid have been shown to have certain cytotoxic effects at high concentrations, capable of damaging cell membranes and mitochondrial function [45]. Therefore, the ratio of toxic to protective components (such as ursodeoxycholic acid) in the mixture is crucial [18]. Second, the effects of bile acids are distinctly dose-dependent; they perform normal functions at physiological concentrations, but excessive concentrations can lead to damage to hepatocytes and intestinal mucosa [12,20]. Finally, the scarcity of natural Bovine Calculus Bovis has led to the development of alternatives such as in vitro cultivated Bovine Calculus Bovis and artificial Bovine Calculus Bovis. Fully assessing the safety and bioequivalence of these products is an important step in ensuring public medication safety. 

Regarding regulatory status, as a traditional TCM component, Bovine Bile Acids currently follow the management frameworks for natural drugs or herbal preparations in various countries. This means that their approval and regulation focus more on historical human use data and the overall efficacy and safety of compound formulations, which is significantly different from the approval pathways for single chemical drugs with clear components and structures. However, as understanding of their active components and mechanisms of action deepens, future development of new drugs based on high-purity bile acid mixtures or specific monomer combinations will face stricter modern pharmaceutical regulatory approval processes [46].

Bovine Bile Acids are bioactive complexes composed of various bile acids, and their unique amphipathic steroid structures serve as the physicochemical basis for their diverse functions, including emulsification and signal transduction. Modern research has preliminarily elucidated some of the molecular mechanisms underlying their core pharmacological activities such as anti-inflammatory, antioxidant, hepatoprotective, and neuroregulatory effects, especially through pathways mediated by nuclear receptors like FXR. In terms of application, they are not only essential components of traditional emergency drugs but also hold a place in modern liver disease treatment. 

However, several key research directions regarding Bovine Bile Acids still need in-depth exploration. It is necessary to further clarify how the various bile acids, bilirubin, and other trace components in Bovine Calculus Bovis interact through complex networks to ultimately produce their macroscopic overall therapeutic effects, and research on their synergistic mechanisms is essential. Second, modern biological techniques such as genomics and proteomics should be employed to precisely identify the molecular targets and downstream signaling pathways of different bile acid components, especially their mechanisms of action in the central nervous system implied by the traditional effect of "calming wind and spasms," for which there are currently no definitive research conclusions. Third, the modernization of the quality control system is the cornerstone for ensuring their safety, efficacy, and controllability, and a dual standard based on multi-component quantification and bioactivity evaluation must be established. Finally, based on their established anti-inflammatory and immunometabolic regulatory effects, there is a need to boldly explore new therapeutic indications, such as their potential applications in metabolic syndrome, neurodegenerative diseases, and autoimmune diseases, and to verify these through rigorous preclinical and clinical studies, which may enable this ancient medicinal treasure to contribute new strength in addressing modern disease challenges.

1. Yu ZJ, Xu Y, Peng W, Liu YJ, Zhang JM, et al. (2020) Calculus bovis: A review of the traditional usages, origin, chemistry, pharmacological activities and toxicology. J Ethnopharmacol 254: 112649.
2. Xu K, Deng B, Jia T, Ren M, Chen H, et al. (2024) A review of the Bovis Calculus's intervention mechanism and clinical application in ischemic stroke. Front Pharmacol 15: 1510779.
3. Kong WJ, Xing X-Y, Xiao X-H, Wang J-B, Zhao Y-L et al. (2012) Multi-component analysis of bile acids in natural Calculus bovis and its substitutes by ultrasound-assisted solid-liquid extraction and UPLC-ELSD. Analyst 137: 5845-5853.
4. Liu Y, Tan P, Liu S, Shi H, Feng X, et al. (2015) A new method for identification of natural, artificial and in vitro cultured Calculus bovis using high-performance liquid chromatography-mass spectrometry. Pharmacogn Mag 11: 304-310.
5. Peng C, Tian J, Lv M, Huang Y, Tian Y, et al. (2014) Development and validation of a sensitive LC-MS-MS method for the simultaneous determination of multicomponent contents in artificial Calculus Bovis. J Chromatogr Sci 52: 128-136.
6. Monte MJ, Marin JJ, Antelo A, Vazquez-Tato J (2009) Bile acids: chemistry, physiology, and pathophysiology. World J Gastroenterol 15: 804-816.
7. Hofmann AF, Hagey LR (2008) Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell Mol Life Sci 65: 2461-2483.
8. Russell DW (2003) The enzymes, regulation, and genetics of bile acid synthesis. Annu Rev Biochem 72: 137-174.
9. Chiang JY (2009) Bile acids: regulation of synthesis. J Lipid Res 50: 1955-1966.
10. Li T, Chiang JY (2014) Chiang, Bile acid signaling in metabolic disease and drug therapy. Pharmacol Rev 66: 948-983.
11. Thomas C, Pellicciari R, Pruzanski M, Auwerx J, Schoonjans K (2008) Targeting bile-acid signalling for metabolic diseases. Nat Rev Drug Discov 7: 678-693.
12. Fiorucci S, Distrutti E (2015) Bile Acid-Activated Receptors, Intestinal Microbiota, and the Treatment of Metabolic Disorders. Trends Mol Med 21: 702-714.
13. Wang H, Chen J, Hollister K, Sowers LC, Forman BM (1999) Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 3: 543-553.
14. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, et al. (1999) Identification of a nuclear receptor for bile acids. Science 284: 1362-1365.
15. Mudaliar S, Henry RR, Sanyal AJ, Morrow L, Marschall HU, et al. (2013) Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease. Gastroenterology 145: 574-582.
16. Jia W, Xie G, Jia W (2018) Bile acid-microbiota crosstalk in gastrointestinal inflammation and carcinogenesis. Nat Rev Gastroenterol Hepatol 15: 111-128.
17. Paumgartner G, Beuers U (2002) Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited. Hepatology 36: 525-531.
18. Beuers U (2006) Drug insight: Mechanisms and sites of action of ursodeoxycholic acid in cholestasis. Nat Clin Pract Gastroenterol Hepatol 3: 318-328.
19. Cabrera D, Arab JP, Arrese M (2019) UDCA, NorUDCA, and TUDCA in Liver Diseases: A Review of Their Mechanisms of Action and Clinical Applications. Handb Exp Pharmacol 256: 237-264.