Abstract
Ejiao, derived from donkey-hide gelatine, is a traditional Chinese medicine with efficacy substantiated by contemporary pharmacological investigations. Chemically, Ejiao contains 19 amino acids, over 2000 peptides, 12 polysaccharides, over 100 volatile components, assorted trace elements, and fatty acids, contributing to its broad therapeutic potential. Quality control methods for Ejiao primarily involve high-performance liquid chromatography, chromatography-mass spectrometry hyphenation, DNA molecular identification, and nuclear magnetic resonance. Pharmacological studies have demonstrated Ejiao's blood-nourishing, anti-anaemic, lung-protective, anti-ageing, anti-fatigue, antioxidant, immune-modulating, anti-tumour, anti-osteoporotic, anti-Alzheimer, oestrogen-mimicking, and skin-repairing effects, with emerging applications in gastrointestinal and cardiovascular diseases. Future research should focus on elucidating its mechanisms of action, such as osteogenesis through Wnt/β-catenin and BMP/Smad pathways, antioxidative effects through Nrf2/HO-1 signalling, and immune modulation via cytokine regulation. Additionally, clinical trials and standardisation efforts are essential to optimise its therapeutic potential.
Introduction
Traditional Chinese medicine (TCM) emphasises harmony between humans and nature. It views health as a balance between the body and its environment. Disruptions to this balance are considered the root cause of disease, and TCM treatments aim to restore harmony while addressing symptoms and underlying causes. 1 A key therapeutic approach in TCM involves using natural products for disease prevention and treatment. Ejiao, also known as Asini Corii Colla, holds a venerable 2000-year history within TCM. This medicinal gelatine, extracted from donkey (Equus asinus) hide in Dong’e county, Shandong, China, has been referenced in early medical texts since the early Tang dynasty. 2 Traditionally employed to arrest bleeding and nourish the blood, Ejiao is frequently used by menstruating women. It is also reputed to enhance the immune system, alleviate symptoms of dizziness and insomnia, and counteract the aging process.3-5 Modern pharmacological experiments have substantiated some of these traditional uses.
The 2020 Pharmacopoeia of the People's Republic of China lists over 150 authorised Ejiao product manufacturers, underscoring its extensive market presence. 4 As global interest in alternative and traditional medicinal products grows, particularly among female consumers seeking cosmeceutical and health benefits, concerns about the purity of Ejiao and its potential adulteration with various animal skins or recycled leather have emerged. 6 Therefore, it is timely to examine the components of Ejiao, its pharmacological effects, and quality control methods to address consumers’ concerns. This article presents a comprehensive review of Ejiao, delving into its constituents, quality control methods, and pharmacological effects, aiming to contribute to contemporary research on this age-old remedy.
Literature Search
An extensive literature search related to Ejiao was conducted in September 2024 using Google Scholar, Web of Science, PubMed, and China National Knowledge Infrastructure (CNKI). Keywords included “Ejiao” “E jiao” “E-jiao” “E’jiao” “Colla Corii Asini” “Asini Corii Colla” “donkey-hide gelatin” and “阿胶” This review focuses on relevant original research articles on Ejiao, with an emphasis on findings from recent years.
Chemical Composition
Proteins, Amino Acids and Peptides
Ejiao is a complex mixture comprising proteins, amino acids, and peptides. 7 In commercially available Ejiao products, protein content typically ranges from 70% to 90%. 8 A shotgun proteomics analysis conducted on donkey skin and bone samples identified 2995 master proteins with high Protein FDR Confidence. 9 The proteins derived from donkey skin exhibit a broad molecular weight distribution, ranging from 10 to 250 kDa. 10 Among these, peptides such as GLPGAPGLR, GAAGIAGPK, GEAGAQGPMGPAGPAGAR, GPTGEPGK, GPTGEPGKPGDK and HGNRGEPGPVGSVGPVGAVGPRGPSGPQGVRGDK have been recognised as species-specific markers. These peptides serve as valuable tools for assessing the quality of animal gelatine products and play a critical role in ensuring the authenticity and traceability of Ejiao.9,11,12 Utilising advanced analytical techniques such as nanoscale liquid chromatography-tandem mass spectrometry (nano LC-Q-Exactive-MS/MS) and shotgun proteomics, 2291 peptides corresponding to 678 Equidae proteins have been identified, providing deeper insights into the composition of Ejiao. 13
Amino acids, the fundamental building blocks of peptides and proteins, serve as crucial quality control indicators for Ejiao. Research has identified 19 amino acids in Ejiao, including nine essential ones, with a notably high content of non-essential amino acids such as glycine (19.24%) and proline (12.27%), while essential amino acids are present in smaller proportions, as detailed in Table 1.7,11,13-17
Amino Acid Composition in Ejiao.
The content in the table represents the average values of common constituents within the literature from different sources.
Glycosaminoglycans
Glycosaminoglycans (GAGs) are distinctive components of the extracellular matrix and interstitium in animal connective tissues. 18 They are classified based on their monosaccharide composition and linkage patterns, including hyaluronic acid (HA), heparin (Hep), chondroitin sulphate (CS), dermatan sulphate (DS), and keratan sulphate (KS). In donkey-hide gelatine, DS was first isolated and characterised in 1994, with detailed analyses of its physicochemical properties. 19 Advanced analysis using liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF) identified heparan sulphate (HS)/heparin in Ejiao, including eight common disaccharides and four rare N-unsubstituted disaccharides with diverse domain structures. 20 Similarly, LC-MS-ITTOF analysis of GAGs, including chondroitin sulphate, dermatan sulphate, and hyaluronic acid in Ejiao, demonstrated significant variations in sulphation patterns, chain lengths, and quantities across different fractions. 21 The polysaccharide content in Ejiao ranges between 26–35 mg/g. 22 with detailed composition data presented in Table 2.
Polysaccharide Composition in Ejiao.
Volatile Composition
Numerous studies have investigated the volatile composition of Ejiao using various analytical methods. One study employed GC-MS, GC-O, sensory analysis, and antioxidant assays to examine the aroma-active components and antioxidant activity of Ejiao from different geographical origins, identifying 23 key aroma-active compounds, highlighting significant geographical clustering, and establishing a strong correlation between antioxidant activity and aroma components. 23 Another study developed and optimised a headspace solid-phase microextraction (HS-SPME) method combined with GC-MS and AMDIS, identifying 41 volatile components in Ejiao, including ten pyrazines, eight aldehydes, five esters, six ketones, and 13 other compounds. 24 Additionally, HS-SPME coupled with GC-MS identified 71 volatile compounds in dissolved Ejiao, predominantly lactones, acids, esters, and heterocyclic compounds such as pyrazines and furan rings. 25
In a separate investigation, 65 volatile substances were identified in Ejiao, primarily consisting of aldehydes, pyrazines, ketones, and sulphur-containing compounds. 26 Additionally, solid-phase microextraction (SPME) and solvent-assisted flavour evaporation (SAFE) were used to extract volatile components, identifying key aroma-active compounds, with pyrazines, acids, and sulphur-containing compounds emerging as the primary contributors to Ejiao's distinctive flavour profile. 27 Another study identified 23 volatile compounds, 12 of which exhibited distinct odours, with methyl isothiocyanate significantly contributing to Ejiao's characteristic aroma. 28 Furthermore, GC-IMS analysis of Ejiao at different processing stages identified 47 volatile flavour compounds, shedding light on their formation mechanisms and providing insights into flavour and quality control. 29 Detailed composition analysis of the volatile composition in Ejiao is presented in Table 3.
Volatile Composition in Ejiao.
Abbreviations: GC-MS, gas chromatography-mass spectrometry; GC-O, gas chromatography-olfactometry; OVA, odour activity value; GC-IMS, gas chromatography-ion mobility spectrometry.
Trace Elements
The name Ejiao originates from the use of water from Ajing in Shandong. 5 Ajing water is rich in magnesium and other trace elements, such as zinc, iron, calcium, and strontium, which are beneficial for human health. 30 Studies using microwave digestion coupled with inductively coupled plasma mass spectrometry (ICP-MS) identified five trace elements in Ejiao: iron, zinc, manganese, barium, and strontium. 31 Studies using microwave digestion coupled with inductively coupled plasma mass spectrometry (ICP-MS) identified five trace elements in Ejiao: iron, zinc, manganese, barium, and strontium. 32
Lipids and Fatty Acids
The crude fat content in Ejiao ranges from 4% to 6%, 33 The primary components are oleic acid, linoleic acid, and palmitic acid, along with trace amounts of plant oils such as arachidic acid. 34 The major fatty acids in Ejiao include methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate. 35 Furthermore, GC analysis of donkey oil has revealed the presence of fatty acids such as linoleic acid and linolenic acid. 36
Quality Control
Chromatography
High-performance liquid chromatography (HPLC) is a key method for determining the amino acid profile of Ejiao. 4 Various studies have explored alternative methods to analyse the amino acid constituents in Ejiao. Automated amino acid analysers were employed to quantify amino acids in different batches of Ejiao.14,16 One approach used a phenyl isothiocyanate column with reversed-phase HPLC, enabling rapid identification of 13 amino acids within 30 min. 15 Another method simultaneously determined L-hydroxyproline, glycine, alanine, and L-proline in Ejiao. 37 Gradient elution techniques, transitioning from micellar to high submicellar liquid chromatography, have also been applied to study the amino acid composition of Ejiao. 38 Additionally, an HPLC fingerprint has been established for enzymatic hydrolysates of Ejiao, demonstrating high reproducibility. 39 Fingerprint profiles were developed to assess fatty acids in donkey bones, skin, and Ejiao, particularly focusing on linoleic acid and γ-linolenic acid, which are important for quality control. 34
Chromatography-Mass Spectrometry Hyphenation
The Chinese Pharmacopoeia 2020 utilises HPLC-MS technology to identify characteristic peptides in Ejiao, ensuring specificity, sensitivity, and precision. 4 However, the method requires a lengthy 12-h enzymatic hydrolysis step, which limits detection efficiency. Recent advancements, such as ultrasound-assisted trypsin digestion, have reduced this process to just 10 min, significantly improving efficiency.40,41 Innovations in chromatography-mass spectrometry, including ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS), 42 nanoscale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS), 43 and ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ/MS), 41 have greatly enhanced Ejiao's quality assessment. UPLC-QQQ/MS has been used to identify equine-derived components in Ejiao,41,44 as well as porcine-derived components. 45 Specific peptides linked to donkey-hide were also detected using this technology, enabling highly specific analysis. 46 A liquid chromatography-tandem mass spectrometry method utilising species-specific marker peptides was developed to detect adulteration in donkey hide gelatine at levels as low as 0.1%. This robust approach provides a reliable method for quality control in medicinal and protein-based materials.12,46 Moreover, Wu et al identified specific peptide markers using LC-QTOF-MS/MS and LC-QQQ-MS/MS, achieving precise and sensitive authentication of Ejiao, with 57 out of 110 commercial products identified as unqualified. 47 These advancements highlight significant progress in improving detection efficiency and authenticity verification for Ejiao products.
Further research confirmed the presence of specific peptides by comparing collagen sequences from various animal skins and detecting these peptides using UPLC-QQQ/MS.48,49 A combined approach of UPLC-MS and metabolomics has been used to analyse differences between Ejiao and related materials, offering promising directions for the quality control of glue-based medicinal substances. 50 One study developed an LC-MRM-MS method targeting marker peptides from bone-specific proteins. This method enables the detection of donkey-hide gelatine adulterated with donkey bone gelatine at levels as low as 50 mg/g. This advancement significantly enhances quality assurance for commercial Ejiao products. 9
One study employed LC-MSⁿ to identify 72 bioactive compounds in Fufang Ejiao Syrup (FES), demonstrating its immune-enhancing effects on bone marrow cells and neuroprotective activities against H2O2-induced oxidative damage in SH-SY5Y and bEnd.3 cells. 51 Another investigation utilised a high-throughput Direct Infusion–Multiple Reaction Monitoring Cubed (DI–MRM³) approach for widely targeted bi-omics of Colla Corii Asini, achieving reliable identification of metabolites and peptides. Notably, nine specific peptides effectively differentiated authentic Ejiao from other gelatine sources. 52
DNA Molecular Identification
The mitochondrial Cytb gene was amplified using PCR and subsequently digested with the restriction enzyme BamH I. This revealed that donkey-derived components in Ejiao could be cleaved into two fragments (76 bp and 314 bp), while horse-derived components remained intact. This PCR-RFLP method effectively distinguished donkey-derived components in Ejiao from horse-derived ones, providing a reliable tool for species identification and quality control. 53 A multiplex PCR method targeting species-specific nuclear and mitochondrial DNA sequences was developed to accurately identify horse, donkey, mule, and hinny hides, as well as horse- and donkey-derived components in Ejiao, offering high specificity with a detection limit of 0.2 ng DNA. 54 However, the high-temperature boiling process during Ejiao preparation can severely damage DNA. To address these challenges, loop-mediated isothermal amplification (LAMP) technology has been utilised for detecting adulteration in Ejiao. A LAMP assay was developed to authenticate Ejiao rapidly and specifically within one hour, demonstrating high sensitivity (10−3 ng). 55 This technique effectively circumvents issues related to DNA damage from boiling or high pressure.
Several advanced methods have been developed for authenticating donkey-derived components in Ejiao and detecting adulterants. One study established a non-toxic, PCR-based approach that achieved high-quality DNA extraction with 100% sequence similarity to GenBank's donkey species. 56 Another utilised COI DNA barcoding with PCR and phylogenetic analysis to efficiently distinguish donkey hides from counterfeit hides of horses, mules, and bovines, achieving over 99% sequence consistency with GenBank data. 57 An optimised TaqMan real-time PCR method, targeting mitochondrial COI genes with highly specific probes and primers, successfully identified adulterants such as horse, ox, and pig DNA, revealing contamination in some market samples. 58 Additionally, a two-step PCR method based on highly repetitive SINE elements has been established for multi-species-specific detection. 59 New species-specific primers have also been developed, achieving a detection sensitivity of 1 ng/μL for horse DNA and 0.1 ng/μL for donkey, cow, and pig DNA. 60
Nuclear Magnetic Resonance (NMR) Technology
Low-field nuclear magnetic resonance (LF-NMR) technology has been employed to authenticate and evaluate the quality of Ejiao, successfully distinguishing various donkey-hide gelatine samples and determining the content of four major amino acids present in the product.61,62 Additionally, a nuclear magnetic resonance (1H-NMR) fingerprint for Ejiao has been established, utilising proton nuclear magnetic resonance spectroscopy. Initial analyses using principal component analysis (PCA) revealed that authentic and counterfeit Ejiao samples could not be effectively differentiated. However, combining PCA with partial least squares discriminant analysis (i-PLS) enabled accurate discrimination between genuine and counterfeit Ejiao samples. 63 Additionally, one study employed 1H-NMR-based metabolomics and correlation analysis to explore metabolic changes in APH-induced anaemic rats treated with Ejiao, identifying key biomarkers such as arginine and aspartate. The study highlighted Ejiao's multi-target therapeutic effects on lipid metabolism, energy balance, gut microbiota, and amino acid pathways. 64
Other Quality Control Methods
Various advanced techniques have been employed to enhance the quality control of Ejiao, ensuring its authenticity and effectiveness. Polyacrylamide gel electrophoresis (PAGE) was used to monitor protein composition dynamics during the processing of donkey hide, tortoise shell, and deer antler, revealing significant molecular weight differences and dispersal patterns influenced by boiling time and additives, providing insights for quality control 65 Near-infrared spectroscopy (NIR), combined with chemometric algorithms such as fingerprinting methods and partial least squares discriminant analysis (PLS-DA), demonstrated its potential by accurately identifying Dong'e Ejiao manufacturers and determining storage times. 66 A synergetic strategy integrating Laser-Induced Breakdown Spectroscopy (LIBS) and NIR with PLS-DA models demonstrated excellent discrimination accuracy in the brand characterisation of Ejiao with data fusion outperforming individual techniques in predictive power and avoiding misclassification. 67 Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy(ATR-FTIR) spectroscopy further distinguished authentic Dong'e Ejiao from counterfeit products by identifying characteristic CH and amide vibrational modes, demonstrating its effectiveness for Chinese medicine identification. 68 Additionally, a rapid and non-destructive method using NIR with Fisher discriminant analysis achieved a 100% recognition rate in differentiating pig skin glue, cowhide glue, and donkey-hide glue. 69
Other innovative approaches include mass spectrometry combined with bioinformatics, which identified two specific peptides, Pep-1 and Pep-2, capable of distinguishing Ejiao from horsehide, mule-hide, and other gelatine products. 11 Terahertz time-domain spectroscopy, integrated with principal component analysis and support vector machines, successfully differentiated gelatine from donkey, bovine and porcine, demonstrating its effectiveness in traditional Chinese medicine applications. 70 Furthermore, microwave digestion combined with ICP-MS enabled precise quantification of 14 elements in Ejiao, including major, trace, and heavy metals, with high precision (RSD 2.0%–5.0%), accuracy (recovery 90.0%–117.1%), and sensitivity. 31 High-sensitivity liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-IT-TOF) also provided insights into Ejiao's biological activities by analysing glycosaminoglycan fractions, identifying both common and rare HS disaccharides with significant structural variations. 20 These methods collectively contribute to a robust quality control framework for Ejiao.
Pharmacological Effects of Ejiao
Blood-Tonifying or Anti-Anaemic Effects
Ejiao, a traditional remedy for blood deficiency, has shown notable blood-tonifying effects in both preclinical models and human studies. Wu et al identified two peptides in Ejiao—peptide 11 (VPGPMGPSGPR) and peptide 16 (APGAPGAPGPVGPGPAGK)—which significantly increased erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colonies in mouse bone marrow in a dose-dependent manner. 71 Additionally, Ejiao peptide-iron chelates have been shown to effectively restore haematological parameters in a mouse model of iron-deficiency anaemia (IDA). 72 In the same study, IDA models were established in ICR mice, which received low, medium, and high doses of Ejiao peptide-iron chelates (1.0, 2.0, and 3.0 mg Fe/kg) daily via intragastric gavage for 4 weeks. The treatment reduced intestinal inflammation, improved gut microbiota composition, and enhanced intestinal health compared to traditional iron sulphate supplements. 73
Additionally, Ejiao has demonstrated notable haematopoietic effects in various preclinical models. In a 5-fluorouracil-induced anaemia mouse model, oral administration of Ejiao (2.5 g/kg/day) for 10 days promoted the proliferation of bone marrow nucleated cells (BMNCs) and haematopoietic stem cells (HSCs), enhanced the differentiation of haematopoietic progenitor cells (HPCs), and regulated key haematopoiesis-related pathways, including ECM-receptor interaction, Wnt, PI3K-Akt, and TGF-beta signalling pathways. 74 Another study using an APH-induced anaemia model in male Sprague-Dawley rats (180-220 g) treated with different doses of Ejiao (1, 2, or 4 g/kg) or Lvjiaobuxue Granules (0.8 g/kg) for 21 days revealed that Ejiao improved anaemia by modulating lipid, energy, gut microbiota, and amino acid metabolism, as evidenced by H-NMR-based metabolomics. 64 In a cyclophosphamide-induced anaemia mouse model, Ejiao active components A and B (50 mg/kg/day) administered for 10 days significantly increased bone marrow mononuclear cell counts, haematopoietic progenitor cell colonies (CFU-GM, BFU-E, CFU-E), CD34 expression, and the proportion of S-phase cells, thereby protecting the bone marrow haematopoietic microenvironment and reducing cyclophosphamide-induced damage. 75 Another study used gradient dosage gavage of Ejiao peptide-iron（EPI）chelates in IDA mice and LC-MS/MS for plasma metabolomic analysis, finding that EPI significantly increased hemogram levels, improved iron bioavailability, and regulated endogenous metabolites to promote haematopoiesis and improve iron homeostasis. 76
In a randomised, double-blinded, placebo-controlled clinical trial involving women with blood deficient symptoms, significant improvements in dizziness symptoms were observed after an eight-week treatment with Ejiao. 77 The placebo group showed a decrease in red blood cell haematocrit and count. In contrast, the reduction in the Ejiao treatment group was notably less, suggesting substantial effects of Ejiao in addressing blood deficiency without evident adverse reactions. Ejiao has also elevated haemoglobin levels in pregnant patients with β-thalassaemia by upregulating specific gene pathways associated with red blood cell membrane stability and lifespan.78,79 Moreover, adjunctive therapeutic effects of Ejiao have been observed in chronic aplastic anaemia. 80 These diverse findings underscore Ejiao's multifaceted potential in addressing various haematological conditions. Table 4 outlines the principal pharmacological effects of Ejiao on blood.
Pharmacological Effects of Ejiao on Haematological Disorders.
Lung Protecting Effects
Ejiao has shown both preventive and therapeutic effects in respiratory disease models. In a lipopolysaccharide-induced acute lung injury (ALI) mouse model, Ejiao, administered in varying doses over a specific period, significantly reduced inflammation and pyroptosis by inhibiting the NF-κB/NLRP3 inflammasome pathway and mitochondrial ROS generation, offering a potential therapeutic strategy for ALI. 81 Supporting these findings, a rat model of lung injury induced by intratracheal instillation of artificial PM2.5 demonstrated that Ejiao, administered over 11 weeks, markedly improved lung function and pathological changes by modulating arginine metabolism and inflammatory pathways. 82 Ejiao has also shown efficacy in a cigarette smoke-induced chronic obstructive pulmonary disease (COPD) rat model. Studies highlighted its ability to inhibit airway inflammation and remodelling by regulating the expressions of MMP-2, MMP-9, and TGF-β1. Ejiao significantly reduced these markers in bronchoalveolar lavage fluid and lung tissue while restoring the balance between Th17 and Treg cells involved in airway inflammation.83-85 Further evidence from a COPD model in rats, established through cigarette smoke exposure (1100 ± 10 mg/m², 90 min/day) for 48 weeks, revealed that oral administration of low, medium, and high doses of Ejiao (1, 2, and 4 g/kg) daily for 28 days improved lung function, reduced emphysema severity, and alleviated lung tissue pathological damage, with the high-dose group showing the most significant effects. 86
Collectively, these studies underscore the promising therapeutic potential of Ejiao in addressing various respiratory ailments. The primary pharmacological effects of Ejiao on the lungs are summarised in Table 5.
Pharmacological Effects of Ejiao on the Lungs.
Anti-Ageing, Anti-Fatigue and Antioxidative Effects
A comprehensive study isolated three Ejiao components with varying molecular weights, emphasising their diverse antioxidative and immunomodulatory effects. Notably, components exceeding ten kDa were identified as key contributors to Ejiao's antioxidative and immunomodulatory activities. 87 Further research demonstrated that Ejiao's alkaline protease hydrolysis products exhibited antioxidative properties by scavenging radicals such as hydroxyl radical, DPPH, and ABTS.88,89 Additionally, an assessment of the antioxidative activity of volatile substances in Ejiao revealed a strong correlation between antioxidative indicators and 23 aromatic active compounds. 23 Nine peptides in Ejiao have also been identified as having DPPH and ABTS radical scavenging activities. 90 Notably, enzymatic hydrolysis using Alcalase 2.4L produced ACC peptides with molecular weights primarily below 5 kDa, significantly enhancing their antioxidative activity by improving ABTS+ and DPPH radical scavenging rates. 91
Research into the anti-ageing properties of Ejiao in d-galactose-induced mice highlighted its capacity to enhance antioxidative functions, eliminate free radicals, and modulate the expression of genes associated with aging. 92 Moreover, studies indicated that Ejiao enzymatic hydrolysates demonstrated superior oxygen radical absorbance capacity compared to cold-water fish skin gelatine. 93 Both small-molecule Ejiao peptides and Ejiao were found to significantly prolong swimming endurance in rats, reducing serum malondialdehyde and lipid peroxide levels, showcasing their anti-fatigue and antioxidative effects. 94 Furthermore, Ejiao demonstrated protective effects on skin cells, accelerating wound healing, stimulating collagen and elastin synthesis, and mitigating oxidative stress effects induced by ultraviolet A. 95
These findings highlight Ejiao's multifaceted antioxidative and immunomodulatory potential, positioning it as a promising agent in combating oxidative stress and age-related processes. Table 6 summarises the pharmacological effects of Ejiao on redox status.
Pharmacological Effects of Ejiao on Redox Status and Fatigue.
Immuno-Regulatory Effects
Ejiao has demonstrated significant immunomodulatory capabilities, enhancing both cellular and humoral immunity. Studies reveal its capacity to increase the population of immune-specific rose-shaped formation cells and strengthen the function of the mononuclear phagocyte system. Notably, Ejiao improved the activity of natural killer cells in the spleens of mice, counteracting immunosuppressive effects induced by hydrocortisone. 96
Further investigations showcased that high-dose small-molecule Ejiao peptides (below 1000 kDa) effectively elevated serum haemolysin levels in mice. Ejiao also amplified delayed-type hypersensitivity reactions and stimulated splenic lymphocyte proliferation, emphasising its ability to fortify both humoral and cellular immunity in murine models. 97 The enzymatic hydrolysates of Ejiao were found to increase thymus and spleen indices while extending swimming endurance in mice. 98 In studies on immunocompromised mice, significant increases in serum haemolysin levels and enhanced populations of T lymphocytes (CD3+), helper, and delayed hypersensitivity T cells (CD3+/CD4+) were observed. Ejiao effectively ameliorated cyclophosphamide-induced immunodeficiency in mice by upregulating TNF-α and GM-CSF expression in serum, as well as TNF-α and GM-CSF mRNA expression in the spleen.99,100
Additionally, a specific 10–30 kDa component of Ejiao was identified as a potent immunomodulatory agent. This component elevated the cellular gene expression of TNF-α, IL-6, iNOS, and TLR4 in mice, enhancing their mRNA content within cells. This effect was associated with its ability to bind TLR4 receptors, triggering the phosphorylation of key proteins such as JNK, ERK, and P38. 101 These findings collectively highlight Ejiao's profound immunomodulatory potential, suggesting its promising role in enhancing immune responses through intricate molecular pathways. Table 7 details the pharmacological effects of Ejiao on the immune system.
Pharmacological Effects of Ejiao on Immunity.
Anti-Tumour Effects
Studies have demonstrated the potential of Ejiao in supporting cancer treatment and alleviating chemotherapy side effects. Research involving advanced-stage cancer patients revealed that supplementing Ejiao alongside chemotherapy improved chemotherapy-induced peripheral blood platelet reduction. This treatment stimulated not only platelet regeneration but also enhanced extramedullary haematopoiesis, indicating Ejiao's potential to mitigate the haematological side effects of chemotherapy. 102
Additionally, experiments in rats showed that oral Ejiao supplementation significantly promoted lymphocyte proliferation induced by mitogens and altered the ratios of various immune cell subsets, suggesting a role in alleviating immunosuppressive effects caused by tumours and radiotherapy. 103 These findings indicate that Ejiao may play a role in alleviating or mitigating the immunosuppressive effects induced by tumours and radiotherapy, thereby supporting immune cell responses against cancer.
In another line of research, specific peptides from digested Ejiao products were screened, leading to the identification of a peptide sequence, ADGVAGPK, which exhibited high binding affinity to the anti-tumour target aminopeptidase N (APN), implying potential anti-tumour activity associated with Ejiao. 104 Furthermore, combining Ejiao products with Ginkgo biloba leaf extract post-alkaline protease hydrolysis showed enhanced inhibitory effects on breast cancer cell lines, suggesting a complementary role in cancer treatment. 88
Anti-Osteoporotic Effects
In vitro studies on rat osteoblasts indicated that while Ejiao did not affect osteoblast proliferation, it significantly influenced their differentiation by enhancing alkaline phosphatase synthesis, suggesting a potential role in bone formation. 105 This differentiation-promoting effect was supported by findings that Ejiao prevented high bone turnover and reduced the number of osteoclasts in ovariectomised rats, implying a preventive effect on postmenopausal osteoporosis. 106
However, other studies exploring the skeletal action of Ejiao found no significant changes in osteocyte number, bone mineralisation, or Wnt signalling markers in ovariectomised rats, highlighting the complexities of Ejiao's impact on bone health. 107 In related research, Ejiao calcium was shown to increase serum calcium and phosphorus levels in rat models of rickets induced by vitamin D deficiency, further supporting its potential role in bone health. 108 Moreover, Ejiao has been linked to enhancing the aggregation and activity of megakaryocytes, promoting the proliferation and synthetic activity of chondrocytes and osteoblasts, and accelerating intramembranous ossification, suggesting its potential therapeutic role in bone healing. 109
In summary, while some studies indicate a positive influence of Ejiao on bone health, particularly in preventing conditions like postmenopausal osteoporosis and aiding bone healing processes. The diverse findings underscore the need for further research to unravel the intricate mechanisms and potential applications of Ejiao in promoting overall bone health. The pharmacological effects of Ejiao on bone are summarised in Table 8.
Pharmacological Effects of Ejiao on Bone.
Anti-Alzheimer Effects
Research has highlighted the potential of Ejiao in mitigating factors associated with Alzheimer's disease. Enzymatic hydrolysates of Ejiao were shown to inhibit acetylcholinesterase activity in nerve-like PC12 cells differentiated by nerve growth factor. This inhibition effectively countered the deterioration of acetylcholinesterase induced by hydrogen peroxide, leading to a reduction in β-amyloid (Aβ) accumulation. 93 Furthermore, a specific peptide sequence, SGLDGAKG, from Ejiao demonstrated a high affinity for binding to anti-Alzheimer's targets such as acetylcholinesterase (AChE) and β-secretase 1 (BACE1), suggesting the potential anti-Alzheimer's activity of Ejiao components. 104 Additionally, Ejiao was found to reduce the number of Aβ-positive cells in the hippocampal CA1 region in ovariectomised mice. Its neuroprotective effects and reduction of Aβ deposition significantly enhanced the learning and memory abilities of these mice. 110 Collectively, these findings contribute to the evidence supporting Ejiao's promising role in addressing neurodegenerative conditions, particularly Alzheimer's disease.
Protective Effects on Female Reproductive System
A systematic review and meta-analysis of randomised controlled trials demonstrated that Chinese herbal prescriptions containing Ejiao or Velvet Antler effectively reduced uterine fibroid volume, improved related symptoms, and lowered oestradiol and progesterone levels in non-menopausal women, with fewer adverse events compared to control groups. 111 Several studies highlighted Ejiao's oestrogen-like effects and potential benefits for female reproductive health. In normal female mice, low (8 g/kg) and high (16 g/kg) doses of Ejiao administered orally for 30 days increased uterine coefficients and serum oestradiol while reducing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. 112 Similarly, ovariectomised mice treated with Ejiao at 4, 8, and 16 g/kg for 30 days showed improved uterine pathology, including thicker endometrium and increased uterine glands, particularly at higher doses. 113 In SD rats, Ejiao at 0.35 and 0.7 g/100 g/day for 48 h post-ovulation enhanced endometrial receptivity by increasing oestrogen receptor and vascular endothelial growth factor expression. 114 In perimenopausal SD rats, 8-week treatment with 80, 160, and 320 mg/kg Ejiao reduced ovarian cell apoptosis by modulating Bax and Bcl-2 expression, delaying ovarian failure. 115
For uterine fibroids, high-dose Ejiao (0.94 g/kg/day) improved uterine pathology, reduced oestrogen levels, enhanced haemorheology, and increased FSH and LH levels in SD rats. 116 Additionally, Ejiao altered the gut microbiota in nude mice with uterine fibroids, increasing Bacteroidetes and decreasing Firmicutes after four weeks. 117 These findings suggest that Ejiao supports reproductive health and hormonal balance. A summary is provided in Table 9.
Pharmacological Effects of Ejiao on the Endometrium.
Skin Protective Effects
A randomised controlled trial observed that Ejiao improved skin moisture and enhanced skin tone, demonstrating a high safety profile with no reported adverse effects. 118 Research on Ejiao aqueous extract showed its ability to suppress atopic dermatitis induced by 2,4-dinitrochlorobenzene in mice and its impact on keratinocytes treated with TNF-α/IFN-γ. The extract inhibited various inflammatory mediators in keratinocytes, regulating key markers and signalling pathways associated with atopic dermatitis (AD). Additional studies demonstrated Ejiao's capacity to inhibit inflammatory mediators, suppress the MAPK/STAT pathway, and alleviate AD-like skin lesions in both mice and keratinocytes.119,120 Furthermore, a specific ratio of Glycyrrhiza uralensis and Ejiao water extracts was identified as the most effective treatment for AD. 121 Another study used in vitro antioxidant capacity evaluation, intracellular oxidation balance analysis, and flow cytometry in a 3D full-thickness skin model to determine the skin protective effects of Ejiao. The study found that the combination of Ejiao and cubilose in a 2:3 ratio enhanced cell resistance to oxidative damage, promoted cellular renewal, and improved skin anti-wrinkle capacity. 122
Other Pharmacological Effects
Ejiao exhibited various pharmacological effects beyond skin protection. Research revealed its antibacterial properties against pathogens like enterohaemorrhagic Escherichia coli and Salmonella typhimurium, showing inhibitory effects on pathogen invasion. 123
Under serum-free conditions, Ejiao was found to suppress inflammatory responses induced by LPS in specific cell types and enhance adiponectin secretion in OP9 adipocytes. 124 Additional studies demonstrated Ejiao's potential in preventing increases in body mass and bone marrow adipocyte numbers while improving skeletal redox status in ovariectomised models. 125 Moreover, Ejiao positively influenced cochlear superoxide dismutase levels in iron-deficient deaf models, showing similar efficacy to iron supplements in improving hearing abilities. 126
Discussion
Ejiao has long been utilised in TCM for its reputed therapeutic benefits. This review provides a comprehensive analysis of the chemical composition, pharmacological properties, quality control measures, and potential avenues for future research, with an emphasis on its use as a health-promoting and medicinal product.
Ejiao is rich in proteins, particularly collagen, which undergoes partial hydrolysis to form bioactive peptides.8,13 These peptides, with a small molecular size, are believed to have the ability to penetrate physiological barriers, enhancing their biological activity.71,90,97,104 The major protein component, collagen, plays a significant role in lung protecting,81,82 blood-tonifying64,71 and skin regeneration. 120 Additionally, these peptides contribute to Ejiao's anti-aging properties.88,89,93,104 and tissue healing effects, 109 as demonstrated by various studies highlighting its role in promoting osteogenesis and bone tissue regeneration,105-107 which are central to Ejiao's therapeutic uses
Ejiao contains GAGs, such as hyaluronic acid and chondroitin sulphate. These compounds regulate the hydration status of bone tissue, which plays a critical role in maintaining bone flexibility, strength, and overall biomechanical properties.127,128 Their immunomodulatory properties also support Ejiao's use as a tonic for general health improvement, highlighting its role in promoting longevity and vitality in TCM. 129
The volatile components of Ejiao, including pyrazines, aldehydes, and sulphur-containing compounds, contribute to its characteristic aroma and flavour.23,26 While these compounds are primarily responsible for the distinct odour of Ejiao, some, like pyrazines, have been shown to possess antioxidant and antimicrobial properties. 130 These actions may enhance the therapeutic effects of Ejiao, contributing to its broad-spectrum health benefits.
Ejiao is also rich in trace elements such as iron, zinc, calcium, and magnesium,31,32 which are essential for maintaining bone density, 131 enhancing blood production, 132 and supports overall metabolic health. 133 These minerals are believed to contribute to Ejiao's effectiveness in promoting haematopoiesis and improving bone mineralisation, aligning with its traditional use for addressing conditions like anaemia.
The presence of fatty acids like oleic, linoleic, and palmitic acids in Ejiao further enhances its medicinal profile.34-36 These fatty acids contribute to the anti-inflammatory and antioxidative properties of Ejiao, making it useful in the management of inflammatory conditions, 134 skin health, 135 and lung protection. 135 Additionally, these components play a role in improving obesity and osteoporosis. 136
The variable quality of Ejiao products stems from inconsistencies in raw materials and manufacturing processes, highlighting the need for robust quality control. Advanced analytical techniques, including HPLC, mass spectrometry, DNA identification, and NMR, have enhanced the profiling of Ejiao's chemical composition and the detection of adulterants. For example, UPLC-QQQ/MS identifies adulteration at levels as low as 0.1%, 46 while DNA-based methods improve species verification even after processing.53,54 Non-destructive tools like ATR-FTIR and near-infrared spectroscopy enable rapid quality assessments. 68 Despite these advancements, variability in active ingredients persists, underscoring the need for standardised markers and more efficient methods to ensure product consistency and authenticity.
Despite its well-documented therapeutic effects, the precise molecular mechanisms underlying Ejiao's bioactive peptides, polysaccharides, and trace elements remain inadequately understood. Elucidating their interactions with key cellular pathways, such as those involved in osteogenesis and immune modulation, is essential to validate their therapeutic potential. While Ejiao holds a prominent place in traditional Chinese medicine, rigorous clinical trials are imperative to establish its efficacy in modern medical practice. In particular, randomised controlled trials focusing on its effects in conditions such as osteoporosis, anaemia, and tissue regeneration are crucial to providing robust evidence supporting its clinical applications.
As the demand for Ejiao is increasing due to the rise of health consciousness and widespread chronic disease, concerns regarding the environmental impact and ethical sourcing of donkey hides have come to the forefront.137,138 The overharvesting of donkeys for their hides has led to significant declines in donkey populations, raising concerns about the potential extinction of certain breeds and disrupting local ecosystems that rely on these animals. 139 To address these challenges, there is an urgent need for sustainable sourcing practices and regulations that balance the demand for Ejiao with the preservation of donkey populations and their habitats. Ethical farming of donkeys, including the implementation of breeding programmes and habitat conservation efforts, can help ensure that both the production of Ejiao and the welfare of these animals are prioritised in tandem. 140
Conclusion
Ejiao is a complex composition rich in proteins, amino acids, peptides, polysaccharides, volatile compounds, trace elements, and fatty acids, making it a valuable traditional Chinese medicine. However, the intricate composition and high market demand pose challenges for quality control. Presently, HPLC and HPLC-MS techniques are employed for monitoring amino acids and peptides, yet there is a growing need for more sensitive and efficient quality control methods.
In modern pharmacological studies, Ejiao has emerged as a substance with diverse beneficial effects. These effects span a range of health conditions, including anaemia, lung injury, aging, fatigue, oxidative stress, immunological dysfunctions, cancers, osteoporosis, Alzheimer's disease, and various female reproductive and dermatological diseases.
While these studies have shed light on the myriad therapeutic properties of Ejiao, further research is imperative to enhance the clinical application of traditional Chinese medicine and its preparations. Additional investigations into the pharmacological activities and clinical effects of Ejiao and its effective constituents are essential. The intricate composition of Ejiao extract requires in-depth exploration across multiple domains before individual compounds extracted from Ejiao can be safely and effectively applied in clinical settings. This underscores the importance of continued research to unlock the full potential of Ejiao in traditional medicine.141–145
Footnotes
Acknowledgments
The authors thank Universiti Kebangsaan Malaysia and Chongqing Chemical Industry Vocational College for their support.
Use of Generative AI in Writing
The authors used ChatGPT version 3.5 (OpenAI, San Francisco, USA) to polish the language of this manuscript, but they are responsible for the content.
Author Contributions/CRediT
Conceptualisation, W.Y. and K.-Y.C.; writing—original draft preparation, C.G., Z.Z., W.Z. and W.Y.; writing—review and editing, S.K.W., T.W. and K.-Y.C.; supervision, K.-Y.C. All authors have read and agreed to the published version of the manuscript.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The researchers are funded via Fundamental Research Grant (FF-2024-298) by Universiti Kebangsaan Malaysia, the Innovation and Entrepreneurship Training Program for college students of Chongqing Chemical Industry Vocational College (HZY202314315044, HZY202314315045, HZY202314315047) and Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN202404512).
Conflicting Interests
The author(s) declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.