Naringenin is the aglycone obtained by hydrolyzing a molecule of glucose and rhamnose from naringin. Its structural formula is shown below, belonging to the dihydroflavonoid compound, which is mainly found in Rutaceae plants such as grapefruit, tomatoes, grapes, and citrus fruits. Domestic and international pharmacological studies have shown that naringenin has various pharmacological activities such as antibacterial, anti-inflammatory, antioxidant, antifibrotic, anticancer, antitumor, antiviral, anti-arrhythmic, antitussive, prevention of atherosclerosis, immune regulation of fat metabolism, protection of liver function, and estrogen-like effects. It can be developed and applied in the fields of medicine, food, and more.

Extraction of Naringenin

Although naringenin can be directly extracted from various raw materials, the extraction yield is relatively low. Currently, the industrial process mainly relies on hydrolyzing naringin to remove glycosyl groups for large-scale production of naringenin. ① Extraction after acid hydrolysis. Before extracting naringenin from raw materials, preprocessing is necessary, primarily to allow naringin to hydrolyze and produce naringenin, which is then extracted. For example, using crude naringin as the raw material, recrystallization can yield highly pure naringin, which can then be hydrolyzed with H2SO4 and extracted with ethyl acetate to obtain naringenin. The acid hydrolysis method is characterized by being inexpensive and simple, and the resulting naringenin has a high purity. However, inorganic acids such as hydrochloric acid and sulfuric acid used in the acid hydrolysis process can easily cause environmental pollution and safety issues, so using food-grade organic acids to replace inorganic acids has become a trend. Compared to sulfuric acid and hydrochloric acid as catalysts, under organic acid catalysis, high purity naringenin can be obtained without the need for recrystallization purification, and the amount of organic solvent required is less. However, the disadvantages are that the naringin hydrolysis rate and naringenin yield are lower. ② Extraction after enzymatic hydrolysis. Using enzymes as catalysts to hydrolyze naringin, pure naringenin can also be obtained after recrystallization. For instance, using cross-linked naringinase aggregates to hydrolyze naringin to produce naringenin, by optimizing factors such as substrate concentration, temperature, pH value, enzyme dosage, and reaction time, the yield of naringenin under optimal process conditions can reach 97.72%. Currently, in addition to the enzymatic hydrolysis of naringin outside the organism to obtain naringenin, it is also possible to use Saccharomyces cerevisiae to express the naringenin-producing gene from Arabidopsis thaliana, directly obtaining naringenin through de novo production within the organism, and then extracting naringenin from it. The enzymatic hydrolysis method is simple but costly, and the hydrolysis process is difficult to control, leading to unstable yields of naringenin. To reduce costs and avoid the complex purification steps of enzymes in the early stages, pure enzymatic hydrolysis is generally not used, and instead, molds that produce naringinase are directly mixed with naringin to hydrolyze naringin.

Structural Derivation of Naringenin

Naringenin is soluble in ethanol, ether, and benzene but practically insoluble in water. Its poor lipid solubility and water solubility can be improved by introducing strongly lipophilic or hydrophilic groups to modify its structure, thereby enhancing its bioavailability. The structural modifications primarily involve alkylation, acylation, sulfonation, glycosylation, and the formation of metal complexes of the hydroxyl groups. Researchers have prepared a large number of naringenin derivatives using the structure of naringenin as the parent nucleus.

① Structural derivation on phenolic hydroxyl groups. The benzene ring of naringenin contains many phenolic hydroxyl groups, which can react with alkyl halides to form ethers. These alkyl halides can be aromatic compounds, aliphatic compounds, and heterocyclic compounds, resulting in a variety of ether derivatives, some of which increase hydrophilicity, some increase lipophilicity, and some are linked to macromolecular substances. At the same time, phenolic hydroxyl groups are also prone to react with carboxylic acids, acid halides, or phosphoryl halides, producing products with various biological activities.

② Structural derivation on the ketone carbonyl group. In the structure of naringenin, there is a ketone carbonyl group, which is also an easily reactive site. It can react with phenylhydrazine, aromatic formylhydrazines, and thiosemicarbazides to form naringenin Schiff bases. It can also react with hydroxylamine hydrochloride, methoxamine hydrochloride, and benzyloxyamine hydrochloride to prepare naringenin oxime derivatives. It can also react with ethylenediamine to form naringenin dimers.

③ Other structural derivations. Naringenin is easily oxidized, including by increasing phenolic hydroxyl groups on the benzene ring and dehydrogenation to produce double bonds. At the same time, due to the electronic effect of hydroxyl groups on the benzene ring, naringenin can participate in some nucleophilic addition reactions. The oxygen-containing heterocyclic ring in naringenin is prone to ring-opening under alkaline conditions, generating α,β-unsaturated ketone derivatives. Moreover, naringenin can be glycosylated to form corresponding glycosides, typically at the phenolic hydroxyl group or on the benzene ring. In addition, the benzene ring is prone to undergo Mannich reactions, generating general Mannich bases or new heterocyclic compounds.

Although naringenin possesses a wide range of pharmacological activities, its clinical applications are limited due to its poor water solubility and lipid solubility, susceptibility to oxidation, poor absorption in the body, and low oral bioavailability. Therefore, researchers have developed various nano-delivery vehicles loaded with naringenin, such as nanoparticles, nanoemulsions, liposomes, and nanosuspensions. These nano-delivery vehicles significantly enhance the stability, solubility, and bioavailability of naringenin, enabling the prevention and treatment of various diseases.

1、Polymer Nanoparticles

Polymer nanoparticles include polymer nanogels, nanocapsules, nanomicelles, and so on. Compared to traditional treatment methods, polymer nanoparticles enhance the biocompatibility, bioavailability, safety, drug permeability, and stability of drugs in drug delivery applications. They also protect drugs from being hydrolyzed by hydrolytic enzymes, thereby improving treatment efficacy. Depending on their composition, polymer nanoparticles can be divided into synthetic polymer nanoparticles and natural polymer nanoparticles.

A variety of synthetic polymers with biocompatibility, degradability, and low toxicity, including polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycolated polylactic acid, have been proven to be suitable for the construction of naringenin nanoparticles. When naringenin is encapsulated using polylactic acid and polyvinyl alcohol, it is found that these nanoparticles exhibit better encapsulation efficiency and sustained release characteristics of naringenin under simulated gastrointestinal conditions, resulting in a 4.7-fold increase in the bioavailability of naringenin. The hydrophilicity and hydrophobicity, as well as targeting properties, of synthetic polymers can affect the structure and properties of polymer nanoparticles. Amphiphilic polymer molecules, such as polyethylene glycolated polylactic acid and methoxypolyethylene glycol-polycaprolactone, can spontaneously form a core-shell structure in aqueous media. Their hydrophobic cores can serve as a reservoir for naringenin, encapsulating the hydrophobic naringenin in the polymer micelles, thereby improving the solubility/dispersibility of naringenin in aqueous media. Hydrophilic polymers, such as dextran and polyvinylpyrrolidone, can encapsulate naringenin in the form of coatings to form nanoparticles. These hydrophilic polymers can form molecular complexes with naringenin, alleviating the crystallization of naringenin and enhancing its solubility and bioavailability. In addition, polycaprolactone is considered to have good targeting properties, able to protect normal non-targeted tissues from toxicity. Therefore, polycaprolactone-naringenin nanoparticles exhibit excellent active targeting effects, enhancing the uptake of naringenin by cancer cells and increasing its toxic effect on cancer cells.

The types of natural polymers used for the construction of naringenin nanoparticles are quite diverse, including proteins (such as silk fibroin, β-lactoglobulin, and lactoglobulin) and polysaccharides (such as chitosan, sodium alginate, and cellulose). There are various methods for constructing natural polymer nanoparticles, mainly including: (1) Cross-linking method: using physical or chemical cross-linkers to achieve cross-linking between natural polymers and naringenin; (2) Top-down method: generating nanoparticles by applying forces such as shearing and impact on natural polymers and naringenin, such as vortex mixing and grinding; (3) Bottom-up method: allowing the assembly of biopolymers and naringenin into nanoparticles through environmental induction (e.g., temperature, pH value, ionic strength, solvent polarity, etc.), such as dissolution-adsorption method and anti-solvent recrystallization method.

2、Lipid Nano-delivery System

In the lipid nano-delivery system of naringenin, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and liposome-based nano-delivery systems are widely used.

Solid lipid nanoparticles (SLNs) are lipid nanoparticles made by encapsulating drugs in lipid cores using natural or synthetic solid lipids such as stearic acid and triacylglycerol as carriers. SLNs have a submicron size (<1000 nm). Compared to polymers, liposomes, and emulsions, SLNs have advantages such as low toxicity, no need for organic solvents, controlled drug release, and low cost. In recent years, SLNs have been widely studied as a naringenin nano-delivery system. For example, NRG-SLNs were prepared using glycerol monooleate (GMO) as the lipid source and polyethylene glycol succinate (TPGS) as the stabilizer through an emulsification homogenization method. Studies have found that naringenin and lipid polymers are miscible, and the lipid nanoparticles exhibit a smooth nanosphere shape with sustained release of naringenin up to 90 hours. NRG-SLNs can effectively reduce liver damage and fibrosis, and SLNs significantly improve the bioavailability and targeted delivery of naringenin to the liver. NRG-SLNs were prepared by emulsification and low-temperature solidification techniques to encapsulate naringenin. NRG-SLNs have a uniform spherical particle structure with a high drug loading capacity and are non-toxic to human lung cancer epithelial cells. The bioavailability of NRG-SLNs is significantly enhanced after administration. A dual-drug SLNs carrier system loaded with naringenin and paclitaxel was prepared using a microemulsion method for the treatment of glioblastoma multiforme (GBM). The results showed that SLNs significantly improved the drug release rate and absorption rate, and improved the targeting of the drugs to GBM.

Nanostructured lipid carriers (NLCs) are the second-generation lipid-based nanoparticles developed based on SLNs. NLCs are lipid-based nanoparticles prepared by adding liquid lipids to solid lipids as a mixed lipid matrix. The characteristics of NLCs lie in their high solubility of drugs in the lipid matrix, high drug loading capacity, controlled drug release, and low burst release of drugs due to their compact structure. By using the emulsion evaporation method combined with low-temperature solidification, naringenin-loaded nanostructured lipid carriers (NRG-NLCs) were prepared. It was found that NRG-NLCs have high drug loading capacity and increase the release rate of naringenin by 3.5 times, promoting the epithelial transport and intestinal absorption of naringenin, and improving the oral bioavailability of naringenin.

Liposomes are spherical lipid vesicles formed by phospholipid bilayer membranes. These lipids are typically natural phospholipids such as soy phosphatidylcholine (SPC), synthetic phospholipids, and dipalmitoylphosphatidylcholine (DPPC). Liposomes can encapsulate both lipophilic and hydrophilic molecules within them, protecting them from degradation and releasing the drug components to a specified target. Research has shown that liposomes can effectively improve and enhance the pharmacological effects of naringenin, such as increasing the solubility and bioavailability of the drug, modifying the drug's properties (particle size, Zeta potential, encapsulation efficiency, and drug release profile), promoting drug targeting, and ultimately improving therapeutic efficacy. Studies have indicated that naringenin liposomes have better physical stability and higher Zeta potential values. Compared to free naringenin, naringenin liposomes exhibit higher solubility and oral bioavailability, with higher drug concentrations in different tissues, especially in the liver.

3、Emulsion-based delivery vehicles

Nanoemulsions are a thermodynamically unstable yet kinetically stable system that are prone to sedimentation, flocculation, and coalescence. Therefore, emulsifiers, typically surfactants and co-surfactants, are often added to nanoemulsions. The latter can adsorb at the oil-water interface, leading to a reduction in interfacial tension, which facilitates the formation of smaller droplets and prevents their aggregation. Particles in nanoemulsions possess a core-shell structure, with a hydrophilic shell composed of amphiphilic materials enclosing a hydrophobic core made of lipophilic materials. Lipophilic materials include various non-polar molecules such as triacylglycerols, diacylglycerols, monoacylglycerols, and bioactive components. The amphiphilic shell comprises various surfactant molecules such as surfactants, phospholipids, proteins, and polysulfides. Research has shown that nanoemulsions can be used as delivery systems for hydrophobic drugs such as naringenin, significantly improving their bioactivity, absorption, and utilization.

Self-emulsifying liquids are anhydrous, homogeneous liquid mixtures composed of oil phase, surfactants, and co-surfactants, representing a concentrated form of anhydrous nanoemulsions. The most significant feature of self-emulsifying liquids is that they rapidly and spontaneously emulsify into nano-sized oil droplets ranging from several nanometers to less than 200 nm in size under the environment of gastrointestinal motility and aqueous gastrointestinal fluids. These nano-sized oil droplets contain drugs dissolved in the oil phase and enhance the interfacial surface area, which can improve the solubilization and permeation properties of drugs. Additionally, self-emulsifying liquids contain high concentrations of surfactants and co-surfactants, which have the advantages of improving drug permeability and drug loading capacity, significantly promoting the bioavailability of highly lipophilic drugs.

4、Nanosuspension Delivery System

Nanosuspension systems can serve as delivery tools for insoluble drugs, reducing cosolvent toxicity, particle size, and enhancing drug solubility, bioavailability, drug loading capacity, and stability. The preparation of nanosuspensions involves the formation of particles, which can be achieved by reducing the particle size of larger crystals to form nanocrystals or by precipitating dissolved molecules to obtain particles. Stabilizers in nanosuspensions play a crucial role in maintaining the stability of nanoparticles, inhibiting spontaneous crystal growth, and maintaining a uniform particle size distribution. The main stabilizers used in preparing nanosuspensions are surfactants or polymers, including Tween-80, Poloxamer 188, polyethylene glycol (PEG), polyvinyl alcohol (PVA), D-α-tocopherol polyethylene glycol succinate (TPGS), and polyvinylpyrrolidone. Currently, many reports have confirmed that nanosuspension systems can significantly improve the physicochemical and pharmacokinetic properties of naringenin. Naringenin nanosuspensions (NRG-NS) were prepared using polyvinylpyrrolidone as a stabilizer. Compared to pure naringenin, NRG-NS has significantly reduced particle size, showing good permeability in the gastrointestinal tract, and significantly improved solubility and bioavailability. Using the precipitation-ultrasonication method, different types of surfactants and polymers, including sodium cholate, sodium dodecyl sulfate, PEG, Tween-80, Poloxamer 188, and TPGS, were used in the preparation of naringenin nanosuspensions. It was found that TPGS has higher emulsification efficiency compared to other surfactants, significantly improving the solubility and oral bioavailability of naringenin. The main reason is that TPGS transforms naringenin from a crystalline state to an amorphous state, promoting its dissolution and reducing naringenin particles from microns to nanometers. 

References

[1] Ji Peng, Zhao Wenming, Yu Tong. Recent Research Progress on Naringenin [J]. Chinese Journal of New Drugs, 2015, 24(12): 1382-1386+1392.

[2] Chen Xixia, Guo Xiaoqian, Yuan Lin, et al. Research Progress in Extraction, Structural Derivation, and Pharmacological Effects of Naringenin [J]. Shandong Chemical Industry, 2023, 52(01): 85-87+90.

[3] Xia Shasha, Wang Qiming, Rao Zhenan, et al. Research Progress on Naringenin and Its Nano-delivery System [J/OL]. Food and Fermentation Industries: 1-11 [2023-09-01].

About the Author

Xiaomichong, a pharmaceutical quality researcher, has been committed to pharmaceutical quality research and drug analysis method validation for a long time. Currently employed by a large domestic pharmaceutical research and development company, she is engaged in drug inspection and analysis as well as method validation.