Storage conditions：Short-term storage at 2~8℃, long-term storage at -20 ~ -80℃

Introduction/Overview

Citric acid anhydrous (CAS No.: 77-92-9), as a widely used natural organic acid, holds an important position in the food industry, pharmaceuticals, cosmetics, and chemical industries due to its excellent acidity and versatility. It is widely used not only as a preservative, acidifier, emulsifier, chelating agent, and buffer but also attracts significant attention from researchers due to its unique role in cell biology and pharmacology research. In recent years, with in-depth research into the biological activity and potential pharmacological mechanisms of anhydrous citric acid, especially its role in apoptosis induction, cell cycle regulation, and oxidative stress-related diseases, its pharmacological value has gradually emerged. This paper aims to systematically review the latest advances in the chemical structure and physicochemical properties of anhydrous citric acid, plant origin and extraction methods, pharmacological activity and mechanism of action, druggability evaluation, and clinical application prospects, providing a theoretical foundation and reference for its research and application in the field of natural product pharmacology.

Chemical structure and physicochemical properties

Anhydrous citric acid is a tricarboxylic acid with the chemical formula C₆H₈O₇ and a molecular weight of 192.1230. Its molecular structure includes three carboxyl groups (-COOH) and one hydroxyl group (-OH), giving it strong acidity and good water solubility. The LogP value of anhydrous citric acid is -0.9049, indicating strong hydrophilicity, water solubility as high as 83.2979 mg/mL, and TPSA (topological polar surface area) of 132.13 Ų, indicating high polarity that makes it difficult to cross the blood-brain barrier (low BBB permeability). Additionally, anhydrous citric acid does not inhibit hERG channels, and the Ames-induced mutagenic test result was zero, indicating high safety and low toxicity risk.

Structurally, the tricarboxyl group structure of anhydrous citric acid gives it excellent chelation ability, allowing it to form stable complexes with various metal ions, which is also an important physicochemical basis for its use as a food additive and industrial acidifier. Its acidic nature makes it a key player in regulating pH and buffering systems.

Plant Origins and Extraction Methods

Citric acid was originally isolated from the fruit of citrus limon, and naturally occurs in various citrus fruits and other plant tissues. Its content is higher in fruits such as lemons, oranges, and grapefruits, mainly present in free form in juice. Industrially, citric acid production mainly relies on fermentation, using microorganisms such as Aspergillus niger to synthesize citric acid by fermenting sugar substrates (such as glucose and sucrose), followed by crystallization and drying to obtain anhydrous citric acid.

Methods for directly extracting citric acid from plants are limited by its content and extraction efficiency, and typically use juice pressing, centrifugation, solvent extraction, and membrane separation techniques. In recent years, the application of ultrasound-assisted extraction and enzymatic hydrolysis technology has improved the extraction efficiency and purity of citric acid, providing a more convenient source of raw materials for natural product research.

Pharmacological activity research

Research on anhydrous citric acid in pharmacology mainly focuses on its regulatory effects on cellular physiological functions, especially in apoptosis, cell cycle regulation, and oxidative stress.

1. Induces apoptosis
   In the human keratinocyte cell line HaCaT, anhydrous citric acid can induce apoptosis, manifesting as changes in cell morphology, upregulation of apoptosis-related protein expression, and increased DNA fragmentation. This action suggests that anhydrous citric acid has potential pharmacological value in regulating cellular fate, especially when applied in pathological skin conditions.

2. Cell cycle block
   Research shows that anhydrous citric acid can block the HaCaT cell cycle during the G2/M and S phases, hindering cell proliferation. This cell cycle regulation may be realized by influencing the expression of cyclins and related kinases, suggesting their potential roles in tumor biology and cell proliferation regulation.

3. Oxidative damage induced
   Anhydrous citric acid causes oxidative damage in the liver by reducing antioxidant enzyme activity (such as superoxide dismutase, glutathione peroxidase, etc.), leading to increased reactive oxygen species (ROS) levels and enhanced cellular oxidative stress. This finding reveals the dual role of anhydrous citric acid in liver metabolism and oxidative stress-related diseases, potentially acting as an inducer in oxidative stress models and highlighting its importance in hepatic toxicology.

4. Other biological functions
   Anhydrous citric acid, due to its acidifying and chelating properties, plays an auxiliary role in regulating microenvironmental pH, metal ion homeostasis, and cellular metabolism. Related research is gradually underway.

Mechanism of action and molecular targets

The biological effects of anhydrous citric acid are closely related to its molecular structure and metabolic pathways. The mechanisms by which it induces apoptosis and cell cycle arrest are not yet fully understood, but studies suggest that the following molecular targets and signaling pathways may be involved:

1. Targets related to metabolic acidosis
   Anhydrous citric acid has a potential link to several key targets associated with metabolic acidosis, including lactate dehydrogenase A (LDHA), carbonic anhydrase 2 (CA2), sodium-citric acid cotransporter (SLC13A3), anion exchange protein (SLC26A6), proton pump ATPase subunit (ATP6V1A), glutamate dehydrogenase 1 (GLUD1), carbamyl phosphate synthase 1 (CPS1), and 3- Hydroxy-3-methylglutaryl-CoA lyase (HMGCL), hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), and mitochondrial glutamate transporter protein (SLC25A13), among others. These targets are involved in intracellular acid-base balance, energy metabolism, and amino acid metabolism, and may be key nodes in anhydrous citric acid regulation of cellular metabolism and acid-base homeostasis.

2. Oxidative stress signaling pathway
   Anhydrous citric acid reduces antioxidant enzyme activity, promotes ROS accumulation, and activates oxidative stress-related signaling pathways such as NF-κB, MAPK, and Nrf2, thereby inducing cell damage and apoptosis. Activation or inhibition of these pathways plays a decisive role in anhydrous citric acid-mediated cytotoxicity.

3. Cell cycle regulator molecules
   Anhydrous citric acid may block the cell cycle process and suppress cell proliferation by regulating the expression of cyclins, cell cycle-dependent kinases (CDKs), and their inhibitory factors (such as p21 and p27).

4. Metal ion chelation and enzyme activity regulation
   Due to its strong chelating ability, anhydrous citric acid can influence the activity of various metal-dependent enzymes, such as metallases and antioxidant enzymes, indirectly regulating cellular metabolism and redox states.

Druggability evaluation and pharmacokinetics

The drug-making parameters of anhydrous citric acid show good water solubility and low lipid solubility, moderate molecular weight, high polarity, and difficulty crossing the blood-brain barrier, indicating it mainly acts on peripheral tissues. It does not inhibit hERG channels, and the Ames test is negative, indicating low cardiotoxicity and mutagenic risk, as well as higher safety.

Pharmacokinetics, citric acid, as an important intermediate in the TCA cycle in the body, has good bioavailability and metabolic stability. It is rapidly absorbed orally, excreted by the kidneys, has a short half-life, and is widely distributed in the body, but is mainly limited to active metabolic organs such as the blood and liver. Its low lipid solubility limits its penetration into the central nervous system, making it suitable for treating diseases related to surrounding tissues.

However, the high polarity and rapid metabolism of citric acid also limit its duration and targeting as a direct drug, requiring structural modification or improved pharmacokinetic properties through carrier systems.

Prospects and outlooks for clinical applications

Anhydrous citric acid, as a highly safe and widely sourced natural organic acid, has broad application prospects in both clinical and industrial fields.

1. Adjunctive treatment for metabolic acidosis
   Since anhydrous citric acid is involved in acid-base balance regulation and metabolic pathways in the body, its application potential in metabolic acidosis is worth further exploration. By regulating related targets such as LDHA and CA2, it may improve acidosis and assist clinical treatment.

2. A potential therapeutic agent for tumors and skin diseases
   The characteristics of anhydrous citric acid in inducing apoptosis and cell cycle arrest suggest its potential value in inhibiting tumor cell proliferation and regulating skin pathological states. In the future, drug combinations or nanocarrier technologies can enhance their targeting and efficacy.

3. Models and treatments for oxidative stress-related diseases
   Its role in inducing oxidative liver damage provides tools for establishing disease models and also suggests its dual role in oxidative stress-related diseases (such as hepatitis, fatty liver, and neurodegenerative diseases), with potential for protection or therapeutic effects through dose regulation in the future.

4. Safety assurance for food and pharmaceutical additives
   As a commonly used food additive and preservative, pharmacological activity studies of anhydrous citric acid help assess its long-term safety and guide its rational use.

Future research should focus on molecular mechanism analysis, structural optimization, and carrier development of anhydrous citric acid to enhance its bioavailability and targeted therapeutic capabilities, while also conducting systematic preclinical and clinical studies to promote its application into clinical applications.

Conclusion

Anhydrous citric acid, as a long-standing and widely used natural product, not only plays an important role in the food industry but is also being increasingly advanced in pharmacological research. Its multiple biological activities in inducing apoptosis, regulating the cell cycle, and regulating oxidative stress reveal its potential application value in metabolic diseases, tumors, and oxidative injury-related diseases. Although its direct application as a drug still faces challenges in pharmacokinetics and targeting, with the continuous elucidation of molecular mechanisms and advances in drug delivery technology, anhydrous citric acid is expected to become an emerging hotspot in pharmacological research and clinical treatment of natural products. Future multidisciplinary interdisciplinary research will provide a solid foundation for the clinical translation of anhydrous citric acid, promoting broader applications in the field of natural product pharmacology.