EF was first recorded in Shen Nong’s Herbal Classic, which records the nature, taste, meridian tropism, and efficacy of EF, laying the foundation for the modern clinical application of EF. Over time, the therapeutic impact of EF has evolved through extensive research. It is pointed out that EF can treat many diseases, such as Jueyin headache, hernia, abdominal pain, beriberi, vomiting blood, acid regurgitation, and diarrhea (Table 2). Concurrently, Miscellaneous Records of Famous Physicians initially documented EF’s mild toxicity, with ongoing research enhancing EF’s toxicology. Illustrated Classic of Materia Medica recorded that EF harms the eyes and hair. Amplified Herbology (Song Dynasty, A.D. 960–1,279) recorded that EF damages the intestines and stomach. Correlation between Materia Medica Companion (Ming Dynasty, A.D. 1,368–1,644) recorded that EF damages the healthy atmosphere. Mouth ulcers, tongue sores, and dizziness caused by excessive consumption of EF are recorded in Compendium of Materia Medica (Ming Dynasty, A.D. 1,368–1,644). The ChP recorded the minor toxicity of EF and stipulated that the dosage of EF was 2–5 g.

EF has been a staple in clinical prescriptions since antiquity (Table 3). Wuzhuyu decoction, named after the monarch medicine EF in Treatise on Febrile Diseases (Dong Han Dynasty, A.D. 25–220), has the effect of warming the middle to replenish deficiency, lowering qi and stopping vomiting. It was the earliest record of the use of EF in clinical treatment. Synopsis of the Golden Chamber (Dong Han Dynasty, A.D. 25–220) mentioned twice the EF of warming channels, dispelling cold, and stopping vomiting, which is an important medicine for tonifying the spleen and stomach. Among the over 5,000 prescriptions in Thousand-Gold Essential Formula for Emergency (Tang Dynasty, A.D. 618–907), there were 143 prescriptions mentioned EF. The Essential Secrets from the Imperial Library (Tang Dynasty, A.D. 618–907) had over 6,000 prescriptions, and the number of prescriptions contained EF reached 176. Formula of Peaceful Beneuolence Pharmacy (Song Dynasty, A.D. 1,078–1,085) was the first official preparation standard. This book recorded a total of 788 prescriptions, and 13 referred to EF. In the clinical realm, essential formulas featuring EF encompass the Wuzhuyu Decoction, Zuojin Pill, Wenjing Decoction, and Sishen Pill, among others.

In addition to examining EF in traditional medical texts, the study of EF in botanical drugs has been extensively explored in contemporary medical settings. Wuzhuyu Decoction can treat chronic non-atrophic gastritis (Hu et al., 2023), chronic migraine (Pan et al., 2015; Nan et al., 2022), alcoholic gastric ulcer (Wang X. et al., 2023), and atherosclerosis (Li C. et al., 2022). Zuojin Pill has pharmacological effects such as anti-tumor (Peng et al., 2015), protection of gastric mucosa, anti-inflammation, anti-ulcer, and so on (Wang et al., 2015; Zhang J. et al., 2022). It can treat bile reflux gastritis (Li Y. Y. et al., 2022) and septic lung injury (Yin et al., 2021). Wenjing Decoction can treat primary dysmenorrhea (Gao et al., 2017) and endometriosis (Huang et al., 2023). Sishen Pill has anti-inflammatory and anti-tumor pharmacological effects (Zhang B. et al., 2024), can treat abdominal pain, diarrhea (Li et al., 2024), irritable bowel syndrome (Zhang X. Y. et al., 2021; Zhao et al., 2024), and insomnia (Wang L. X. et al., 2023). EF is recommended as the primary treatment in Japan for ailments like cold headaches, dysmenorrhea, and inflammatory pain in rheumatoid joints (https://www.mhlw.go.jp/index.html). Certain EF-containing prescriptions, including Changkang Tablets, Huatuo Zaizao Wan, Compound Berberine Tablets, and Jiawei Zuo Jin Wan, have undergone extensive research and clinical application. Changkang Tablets are used in the treatment of dysentery, abdominal pain, and tenesmus (Shi et al., 2013; Guan et al., 2020), Huatuo Zaizao Wan can treat Alzheimer’s disease (Jiang et al., 2023) and stroke (Duan et al., 2017).

Alkaloids are the metabolites of EF, mainly composed of indoles (Supplementary Figure S1) and quinolones (Supplementary Figure S2). Alkaloids fundamentally possess a circular form and are non-soluble in water, resulting in a higher concentration of EF alkaloids in ethanol. Indoles are synthesized mainly through methanesulfonic acid and amino acids. Over ten different compounds, including evodiamine, rutaecarpine, and dehydroevodiamine, were extracted from EF (Zuo et al., 2000; Wang Q. Z. et al., 2010; Wang T. Y. et al., 2010; Wang X. X. et al., 2013; Li et al., 2014; Zhao N. et al., 2015; Ma et al., 2021; Qin et al., 2021; Zhao X. M. et al., 2021). Evodiamine and rutaecarpine are the most important metabolites, and their contents are also the highest in EF (Huang et al., 2019; Li D. W. et al., 2020). Dihydroevocarpine and evocarpine are the crucial metabolites of quinolones found in EF (Li Y. H. et al., 2020). Furthermore, the group includes quinolines, organic amines, acridone, and purines (Minh et al., 2003; Kim et al., 2022) (Supplementary Figure S3). Research revealed a reduction in the levels of evodiamine, rutaecarpine, and carpine in EF correlating with the fruit’s diminution. Some commercial evodiamine and rutaecarpine in SEF are below the content specified in ChP (Cao et al., 2019).

Typically, terpenoids (Supplementary Figure S4) originate from methylpentanedioic acid, with isoprene forming the fundamental structural component of the molecular framework (Zhao H. et al., 2021; Qian et al., 2014). Limonin is an oxidized tetracyclic triterpene with a distinctive furan ring, and it is a terpenoid extracted and recognized from EF. They constitute the material basis of the bitter properties of EF (Bae et al., 2020). Its representative metabolites are limonin and rutaevine, and limonin is another index metabolite of EF. In addition, there are high contents of metabolites, such as evodol, obacunone, rutaevine acetate, 6β-acetoxy-5-epillimonin, jangomolide, and shihulimonin A (Lacroix et al., 2011). Due to their significant solubility in fats, terpenoids are typically processed using an ethanol solvent. Research indicates that limonin levels in SEF and MEF are notably elevated, approximately 0.74% and 0.65%, respectively, in contrast to LEF’s mere 0.24% limonin content (Zhang et al., 2021c).

Flavonoids (Supplementary Figure S5) generally refer to a series of metabolites formed by connecting two benzene rings with three carbon atoms. EF additionally has a higher content of flavonoids. It mainly includes these three metabolites: flavonoids and their glycosides, flavonols and their glycosides, and flavonones and their glycosides. Flavonoids and their glycosides are predominantly associated with O-glucose, O-xylose, O-galactose, O-rhamnose, O-rue sugar, and O-mulberry disaccharide. Flavonols and their glycosides mainly include quercetin, isorhamnetin, limocitrin glycosides, and aglycones (Xiao et al., 2023; He et al., 2024). Flavonones and their glycosides are phellodensin F, catechin, and hesperidin, respectively (Zhao Z. et al., 2015; Li and Wang, 2020).

EF has a strong and fragrant smell because of its high content of volatile oil (Supplementary Figure S6). The volatile oil metabolites are monoterpene, sesquiterpene, aliphatic, and aromatic. Within the isolated volatile oil, the proportions of sesquiterpenes exceed 38%, monoterpenes surpass 35%, and esters exceed 13% (Liu S. S. et al., 2019). Furthermore, while EF volatile oil exhibits significant pharmacological properties, it simultaneously constitutes a toxic metabolite of EF. Its metabolites are monoterpenoids, such as myrcene and (E)-ocimene, and sesquiterpenes, such as β-caryophyllene and β-elemene.

EF also contains some organic acids (Supplementary Figure S7), including caffeic acid, citric acid, isocitric acid, trans-caffeoylgluconic acid, and feruloylgluconic acid. EF also contains phenylpropanoids, mainly divided into simple phenylpropanoids, coumarins, and lignans, most of which belong to simple phenylpropanoids. Simple phenylpropanoids include p-hydroxycinnamic acid, ferulic acid, coniferin, chlorogenic acid, etc. Besides the metabolites mentioned above, EF also contains anthraquinones, such as chrysophanol, emodin, and physcion, and steroids, such as β-sitosterol, β-daucosterol, and β-stigmasterol (Supplementary Figure S8).

Processing is the essence of TCM application, which can increase the efficacy and reduce the toxicity of drugs. EF’s processing boasts an extensive historical background, with raw EF typically exerting a significant influence in heating the spleen and eliminating cold. The long traditional technology of processing EF with licorice is pointed out in the Synopsis of the Golden Chamber. Master Lei’s Treatise on Drug Processing (Northern and Southern Dynasties, A.D. 420–479) recorded that EF is processed with salt to enhance the analgesic effect and vinegar to correct the taste. Materia Medica for Dietotherapy (Tang Dynasties, A.D. 618–907) recorded that EF could enhance the antiemetic effect after processing ginger and the analgesic effect after being processed with yellow rice wine. General Records of Holy Universal Relief (Song Dynasties, A.D. 1,078–1,085) recorded that EF can reduce toxicity when processed with soybean products. Prescriptions for Universal Relief (Ming Dynasty, A.D. 1,368–1,644) recorded that EF fried with Psoraleae Fructus can enhance the antidiarrheal effect. Wonderful Well-Tried Recipes (Ming Dynasty, A.D. 1,368–1,644) recorded that EF processed with Coptidis Rhizoma can enhance the antiemetic effect. The process and method of processing EF with licorice are described in detail in ChP (Figure 3). In addition, different doses of licorice can affect EF’s chemical composition and pharmacological effects (Xiao et al., 2012). Certain academics have experimented with varying the EF to licorice dosage ratios, discovering the optimal 100:6.

In its extended clinical application, TCM has developed distinct theories and techniques for detoxifying and improving its healing impact, encompassing both processing and compatibility (Li R. L. et al., 2021). Special processing techniques such as licorice, salt, ginger, and vinegar can reduce the toxicity of EF. The ChP stipulates that EF ought to be fried alongside licorice. Some scholars have found that processing EF with licorice can reduce the toxicity of alkaloids (Ren et al., 2023a). The cytochrome P450 (P450, CYP) enzyme activates evodiamine in EF during metabolism, which leads to liver injury and inflammation. Licorice can reduce the toxicity of EF by inhibiting the P450 enzyme and blocking the metabolic activation of EF (Ren et al., 2024). Licorice can obstruct EF protein coupling by suppressing the P450 enzyme, elevating GSH levels in human liver cells, and mitigating the GSH reduction induced by EF (Ren et al., 2023b). Salt-processed EF can introduce drugs into the kidney channel, reduce toxicity, and ensure the safety of clinical drug use (Hou et al., 2023). When EF is combined with ginger, its antiemetic properties can be amplified. Some studies found that EF processed with ginger, licorice, and salt had better antitoxic effects (Zhang M. et al., 2021). Other studies found that the three processing methods of ginger, licorice, and vinegar could reduce the content of rutaecarpine in EF, with vinegar processing increasing the content of evodiamine, and licorice processing had the most significant effect on reducing toxicity (Li H. et al., 2021). The metabolites of EF obtained by different processing methods, such as stir-frying, roasting, and steaming, are also different (Xiao et al., 2023). A comparative study of various EF-processed products revealed that the combined amounts of evodiamine, rutaecarpine, and evodol in EF for stir-frying exceed those in baking and cooking.

EF combined with other drugs can also counteract its toxicity. For example, the toxicity of EF significantly decreased when EF was used in combination with licorice and jujube (He et al., 2024). EF with Coptidis Rhizoma can enhance the anti-inflammatory effect and inhibit the inflammatory reaction in RAW264.7 cells by significantly reducing the levels of IL-6, TNF-α, and IL-1β (Wang J. et al., 2024). Additionally, it is capable of markedly reducing apoptosis and enhancing the defensive role of the gastric mucosa through the suppression of gastric acid release (Zhang Z. et al., 2024). Berberine is an important metabolite of Coptidis Rhizoma, which can counteract the side effects of evodiamine and reduce the risk of evodiamine in treating gastric cancer (Shi et al., 2013). The combination of berberine and evodiamine can synergistically inhibit the proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis (Du et al., 2017). The researchers found that EF combined with ginger, Citri Reticulatae Pericarpium, Paeoniae Radix Alba, and Angelicae Sinensis Radix can improve the efficacy of EF (Wu et al., 2021).

With the continuous updating of the edition of ChP, the identification, content evaluation, testing technology, and quality standards of EF are constantly improving (Table 4). EF was first recorded in the 1963 edition of ChP, and it was clearly recorded that EF was processed with licorice. Then, the identification method of EF appeared for the first time in the 1973 edition of ChP. In the 1985 edition of ChP, the EF identification method was officially determined as hydrochloric acid filtration, potassium mercuric iodide de-precipitation, and the formation of a reddish-brown ring zone at the interface between dimethylaminobenzaldehyde and EF solution. The ChP has been improving the quality control of EF since 2000. For the first time, the determination method and the lowest value of EF appeared in the 2000 edition of ChP, which stipulates that the total amount of evodiamine and rutaecarpine should not be less than 0.2%. Next, the total amount of evodiamine and rutaecarpine should not be less than 0.15% in the 2005 edition of ChP. Limonin content is adjusted to no less than 1.0% in the 2010 edition of ChP. Limonin content is again adjusted to no less than 0.20% in the 2015 edition of ChP. The 2015 edition of ChP is basically consistent with the 2020 edition of ChP, indicating that the metabolites of EF are relatively stable and can better represent the efficacy of EF. However, the maximum reference dose of metabolites is not specified in ChP, and the toxicity of EF has not been reasonably controlled.

TCM’s effectiveness can differ significantly based on the type of plant, its source, and yield. Diverse environmental factors like soil, air quality, precipitation, and sunlight play a crucial role in shaping EF growth, with each EF metabolite varying in content across regions (Liu Y. et al., 2019). Some studies have shown that the metabolite of EF varies greatly in different locations, and there is little change in the metabolite of EF in different years in the same location (Huang et al., 2008; Zhou et al., 2010). By comparing the EF of different batches of LEF, MEF, and SEF, it was found that the limonin content in SEF was higher than that of MEF and LEF, while the alkaloid content was higher in MEF and LEF. The toxicity of EF is related to the content ratio of limonin and alkaloid. The higher the ratio, the lower the toxicity of EF. In other words, MEF is more toxic than other categories, and SEF is less toxic than other categories (Zhang et al., 2021b). Furthermore, certain academics analyzed the four vital metabolites of ER, ERO, and ERB, namely evodiamine, evodiamine, dehydroevodiamine, and narcissoside, discovering comparable levels of dehydroevodiamine. The content of evodiamine, rutaecarpine, and narcissoside was the highest in ERO and the lowest in ERB (Li C. H. et al., 2021).

The ChP stipulated the detection method of EF and pointed out that evodiamine, rutaecarpine, and limonin are the crucial metabolites of EF. However, these three metabolites do not represent the overall pharmacological effects of EF. Consequently, sophisticated detection techniques are essential for qualitative and quantitative analysis of metabolites in EF. The leading analytical technologies include TLC, HPLC, HPLC-MS, GC-MS, CE, and CCC (Xia et al., 2023). At present, there are many studies on the metabolites of EF. Scholars have isolated three metabolites from EF: rutaecarpine, evodiamine, and evodiamide (Zhou et al., 2006). Then, two new-lactone derivatives, evodinoids A and B, and a new volatile oil, are separated from EF (Xin et al., 2022). In addition, five metabolites of EF were found. They are limonin, 1-methyl-2-undecyl-4(1H) quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine (Zhang et al., 2013). It has been proven that evodiamine is closely related to the hepatotoxicity of EF (Zhang et al., 2021b). Limonin serves as the primary liver-protective metabolite of EF, while evodiamine is the chief liver-damaging metabolite of EF. Subsequently, eleven critical metabolites of EF underwent analysis using non-specific metabonomics and in vitro functional techniques (Yong et al., 2024). Up to this point, a total of 17 metabolites have undergone screening from EF, including neochlorogenic acid, caffeic acid, chlorogenic acid, 3-O-feruloylquinic acid, hyperoside, quercetin-3-O-sambubioside, rutin, dehydroevodiamine, isorhamnetin-3-O-β-D-galactoside, narcissin, isorhamnetin-3-O-β-D-glucopyranoside, diosmin, rutaevine, limonin, evodiamine, rutaecarpine, and evocarpine.

EF has a cardiovascular protective effect, and its aqueous extract can contract the aorta (Hibino et al., 2009b). In managing cardiovascular conditions, evodiamine, rutaecarpine, and limonin serve as crucial metabolites of EF, offering protection against myocardial ischemia-reperfusion (I/R), anti-myocardial fibrosis, anti-arrhythmia, safeguarding vascular endothelial damage, altering vascular tension, and so on. Studies have shown that evodiamine has an anti-atherosclerotic effect, can regulate energy by inhibiting the expression of the β1-adrenergic receptor, and prevents cardiac I/R injury (Xue et al., 2015). Subsequently, evodiamine has the ability to control the growth and movement of vascular smooth muscle cells by blocking the PI3K/AKT axis activation in the traditional route, thereby preventing atherosclerosis onset and progression (Zha et al., 2023). What’s more, evodiamine can prevent isoproterenol-induced cardiac fibrosis by regulating endothelial-to-mesenchymal transition (Huang et al., 2017). Moreover, rutaecarpine promotes endothelial nitric oxide synthase (eNOS) phosphorylation and NO synthesis via the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and calmodulin-dependent protein kinase kinase β (CaMKKβ)/AMP-activated protein kinase (AMPK) signaling pathways through transient receptor potential vanilloid type 1 (TRPV1), and effectively prevent endothelial dysfunction (Lee et al., 2021). Rutaecarpine can also reduce the damage to myocardial cells caused by myocardial infarction by enhancing vascular smooth muscle calcification (Zhan et al., 2021). Some studies have shown that limonin can inhibit adriamycin-induced cardiotoxicity by activating Nrf2 and SIRT2 signal pathways (Li X. H. et al., 2022). Limonin can also inhibit the ubiquitination and degradation of SIRT6, stabilize the level of SIRT6 protein, promote its expression, reduce cardiac hypertrophy, and improve cardiac function (Liu et al., 2022).

EF is an effective botanical drug for treating gastrointestinal diseases, especially evodiamine, rutaecarpine, and dehydroevodiamine (Chen et al., 2023). Evodiamine can act as an antioxidant by blocking the Rho/NF-κB pathway and alleviating gastric mucosal injury (Zhao Z. et al., 2015). It can also inhibit gastritis caused by Helicobacter pylori infection by inhibiting the NF-κB pathway (Yang et al., 2021). Then, evodiamine can effectively improve the imbalance of intestinal microflora and relieve the symptoms of ulcerative colitis by increasing the level of lactobacillus acidophilus and the production of acetate (Wang et al., 2020). In addition, it can suppress gastrointestinal hyperactivity caused by stress via cholecystokinin (CCK) and the CCK1 receptor (Ren et al., 2018). Moreover, rutaecarpine is effective in mitigating gastric damage caused by ethanol through the suppression of NF-κB pathway anti-inflammation, Nrf2 pathway antioxidation, stimulation of Bcl-2, suppression of Bax and caspase-3 expression, and prevention of gastric cell apoptosis (Ren et al., 2020). Some studies have shown that TRPV1/calcitonin gene-related peptide (CGRP) pathway is an important therapeutic target for gastric mucosal injury (Luo et al., 2013). Rutaecarpine can stimulate the TRPV1 receptor to release CGRP, inhibit the excessive secretion of gastric acid, and improve the symptoms of gastric ulcers (Liu et al., 2008). Moreover, Dehydroevodiamine can reduce the inflammatory injury of gastric mucosa by reducing the ERK/p38 signal pathway, down-regulating the expression of myeloperoxidase (MPO), TNF-α and IL-6, upregulating the expression of IL-10, regulating gastric pH and mucosal thickness (Wei et al., 2021). Then, dehydroevodiamine can also improve MNNG-induced gastric mucosal injury and GES-1 migration in chronic atrophic gastritis rats and treat atrophic gastritis by inhibiting hypoxia-inducible factor 1α/vascular endothelial growth factor angiogenesis pathway (Wen et al., 2021). Furthermore, EF polysaccharides have a protective effect on gastric mucosa and can alleviate the symptoms of gastric ulcers. By increasing the expression of Nrf2 and HO-1 protein, reducing the expression of Keap1 protein, activating Keap1/Nrf2/HO-1 signal pathway, and reducing oxidative stress in the stomach (Luo et al., 2023).

The application of EF in neurological disorders is becoming increasingly widespread. EF and its metabolites have neuroprotective effects on neurodegenerative diseases such as ischemic injury, neuropathic pain, neuroinflammation, Alzheimer’s disease (AD), and so on. Studies have shown that EF methanol extract (200 mg/kg) protects neurons and prevents ischemia-induced cognitive impairment (Lee et al., 2011). Then, evodiamine can reduce peripheral hypersensitivity and anxiety in nerve-injured mice (Zhang et al., 2020). Evodiamine can also repair memory and cognitive impairment, protect neurons in vitro, and inhibit glial cell activation and neuroinflammation (Lima and Hamerski, 2019). In the experimental AD mice induced by intracerebroventricular injection of streptomycin, evodiamine (50 and 100 mg/kg) was orally given daily for 21 days, the ability to recognize new targets and the score of water maze test was improved in AD mice (Wang et al., 2018a). In addition, evodiamine can improve AD mice’s learning and cognitive impairment (Wan et al., 2024) and treat AD through antioxidation and anti-apoptosis (Zhang et al., 2018). Moreover, rutaecarpine ameliorates neuronal injury in rats with cerebral I/R by regulating the expression of ERK1/2 and Nrf2/HO-1 pathway (Han et al., 2019). Rutaecarpine can affect Ca²⁺ influx and activate PI3K/AKT signal pathway by activating specific capsaicin receptor TRPV1, inhibit intracellular oxidative stress and apoptosis protease activity, and protect neurons from apoptosis induced by hypoxia-reoxygenation (Yang Y. et al., 2018). Additional research has shown that limonin and its variants are versatile in combating neuroinflammation and neuronal apoptosis by activating PI3K/AKT and reducing TLR4/NF-κB pathway activity and are effective in treating AD (Panda S. P. et al., 2024). Limonin plays a neuroprotective role by inhibiting neuronal autophagy and microglial activation in rats injected with 6-hydroxydopamine (Gao et al., 2023). Furthermore, dehydroevodiamine (10 mg/kg) can improve the symptoms of memory impairment induced by scopolamine in mice. Dehydroevodiamine has a strong protective effect on cognitive impairment through its antioxidant activity, inhibition of neurotoxicity, and intracellular calcium. Therefore, Dehydroevodiamine may be an important drug for treating memory disorders (Shin et al., 2017). Tg2576-induced AD mice were treated with dehydroevodiamine (0.5 mg/kg) for 4 months, which improved the memory impairment of Tg mice and decreased the levels of soluble amyloid-β 40 (Aβ40), soluble Aβ42 and total Aβ peptide in the cortex of Tg mice. Dehydroevodiamine can inhibit the activity of β-secretase in a dose-dependent manner, which is related to the production of Aβ and the formation of neuritis plaques. Dehydroevodiamine may have a therapeutic effect on AD as a β-secretase inhibitor (Shin et al., 2016).

The anti-inflammatory activity of EF has been widely recognized. Research indicates that the 70% ethanol extract of EF can inhibit the inflammatory response in HaCaT cells. It exerts its anti-inflammatory effects by modulating the JAK-STAT and MAPK signaling pathways. This regulation suppresses inflammatory mediators, cytokines, and chemokines, alleviating symptoms associated with atopic dermatitis (Jin et al., 2024). EF has a potent anti-inflammatory and uric acid-lowering effect. The EF water extract can significantly improve the production of serum inflammatory cytokines IL-1β and TNF-α and inhibit the activation of renal NLRP3 inflammatory signal (Wang Z. et al., 2024). In addition, rutaecarpine can significantly reduce the inflammatory response induced by pseudotype severe acute respiratory syndrome coronavirus 2 by blocking the activity of 3C-like protease (Lin et al., 2023). It has been proved that rutaecarpine can inhibit inflammation by inhibiting the NF-κB signal pathway mediated by PI3K/AKT and MAPK and reduce lipopolysaccharide (LPS)-induced cell migration and number by inhibiting Src/FAK pathway (Jayakumar et al., 2021). Rutaecarpine has also reduced inflammatory responses by downregulating interferon-α, IL-23 p19, and IL-17A protein. This anti-inflammatory effect is mediated through the NF-κB and TLR7 signaling pathways (Li Y. et al., 2019). Moreover, evodiamine is anti-inflammatory by inhibiting IL-1β, IL-2, IL-6, IL-8, TNF-α, and other inflammatory factors mediated by NF-κB (Zhang Y. et al., 2022). Evodiamine can also significantly reduce the pathological damage of breast tissue, inhibit the activation of inflammation-related pathways such as AKT, NF-κB p65, ERK1/2, p38, and JNK, and significantly reduce the production of pro-inflammatory cytokines (Yang Y. et al., 2022). Evodiamine can also improve ulcerative colitis by down-regulating NF-κB signal pathway and NLRP3 inflammatory bodies (Shen et al., 2019). Furthermore, limonin can reduce hepatic steatosis, lipid accumulation, and the expression of p-STAT3/STAT3, caspase-8, and prostaglandin-endoperoxide synthase 2, and improve the inflammatory response of non-alcoholic fatty liver (Wang W. et al., 2023). Limonin can effectively regulate the inflammation mediated by CD4⁺T cells and inhibit the proliferation of CD4⁺T cells by inhibiting the nuclear translocation of NF-κB p65 in activated CD4⁺T cells (Kim W. et al., 2009). Limonin also participates in the regulation of inflammatory pathways by effectively inhibiting p38 MAP. Limonin can also counteract hypertension and vascular damage associated with metabolic syndrome by reducing inflammation and fibrosis (Hassan et al., 2018). Additionally, limonin significantly decreased TNF-α, IL-1β, and IL-6 and inhibited the expression of inflammatory factors in lipopolysaccharide LPS-induced acute lung injury in mice (Wang et al., 2018b).

The analgesic effect of EF is closely related to its anti-inflammatory effect. Oral 50% or 70% methanol extract of 200 mg/kg EF has an analgesic effect on writhing induced by acetic acid (Matsuda et al., 1997). The analgesic effect of EF is related to its metabolites, including evodiamine, rutaecarpine, dehydroevodiamine, rutin, and limonin. Evodiamine exerts analgesic effects through various mechanisms, such as inhibiting ion channels, directly suppressing pain signals, reducing neuronal inflammation, restoring the balance between excitatory and inhibitory neurotransmission, and modulating neurotransmitter release (Jiang et al., 2022). In vitro, evodiamine can significantly reduce capsaicin-induced current and thermal hyperalgesia in rats by activating TRPV1 and neuron desensitization (Iwaoka et al., 2016). Evodiamine can also inhibit migraine-like pain response, which may be due to the regulation of nNOS and the inhibition of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor glutamate A1 (Lin et al., 2020). Evodiamine has been shown to inhibit neuropathic pain, improving paclitaxel-induced neuropathic pain by suppressing inflammatory responses and maintaining mitochondrial antioxidant function (Wu and Chen, 2019). In addition, limonin exhibits significant pain-relieving effects at 30 or 100 mg/kg doses. This analgesic effect is likely associated with its anti-inflammatory properties (Matsuda et al., 1998).

EF exhibits potent anti-cancer properties and significant healing properties against various cancers, including lung, liver, stomach, breast, and cervical. Studies have shown that 70% ethanol extract of EF can significantly reduce the vitality of human cervical cancer HeLa cells at 20–60 μg/mL and show a certain concentration correlation (Park et al., 2017). Evodiamine has anti-tumor effects by inducing apoptosis, blocking the cell cycle, regulating autophagy, and inhibiting tumor cell metastasis (Luo et al., 2021; Panda et al., 2023). Evodiamine can treat non-small cell lung cancer (NSCLC) by down-regulating the expression of SOX-9 and β-catenin and significantly inhibiting cell migration by inhibiting epithelial-mesenchymal transition (EMT) (Panda M. et al., 2024). Then, evodiamine can induce cell cycle arrest in the G2/M phase, inhibit cell migration, and inhibit the Notch3 signal pathway against NSCLC by inhibiting γ-secretase (Yang X. et al., 2020). Evodiamine can increase the expression of cleaved-caspase-3, decrease the activity of TSGF and alpha-fetoprotein, induce AKT-mediated apoptosis, and exert an anti-hepatoma effect (Yang F. et al., 2017). Moreover, evodiamine inhibits PI3K/AKT, ERK1/2, and p38 MAPKs and promotes apoptosis of ovarian cancer cells by activating caspase-9/8/3 and poly ADP-ribose polymerase cleavage (Wei et al., 2016). Evodiamine activates retinoblastoma protein through p53 and p21, which selectively inhibits breast cancer stem cells in the G1/S phase, resulting in cancer cell death (Han et al., 2016). Evodiamine inhibits the proliferation and induces apoptosis of cholangiocarcinoma cells, inhibits the migration and invasion of cholangiocarcinoma cells, suppresses IL-6/STAT3 signal transduction by upregulating the expression of SHP-2, and treats cholangiocarcinoma (Zhu et al., 2019).

Beyond that, rutaecarpine can inhibit CYP1A1 and exert an anti-tumor effect by inhibiting the binding of 2,3,7,8-Tetrachlorodibenzo-p-dioxin to its receptor (Rannug et al., 1992). Rutaecarpine can also significantly inhibit human CYP1A2 and CYP3A4 (Don et al., 2003; Iwata et al., 2005). Moreover, limonin can inhibit the cell activity of colorectal cancer cells, block STAT3 signal transduction, and inhibit the proliferation, migration, invasion, and colony formation of colorectal cancer cells (Zhang W. F. et al., 2024). Limonin exhibits properties that combat breast cancer. It has been reported that limonin has cytotoxicity on estrogen receptor-positive or negative human breast cancer cells, which may inhibit proliferation by activating caspase-7 dependent pathway (Kim et al., 2013). What’s more, dehydroevodiamine pancreatic is the activator of DNA damage-inducible transcript 3 (DDIT3) and has the ability to inhibit the AKT/mammalian target of rapamycin (mTOR) pathway. It can effectively inhibit the proliferation of pancreatic ductal adenocarcinoma cells, and the growth of prostate cancer stem cells in vitro and in vivo (Zhu et al., 2024).

EF can warm the stomach, invigorate the spleen, and be used in glucose and lipid metabolism. In fat metabolism, evodiamine reduced the food intake rate and weight gain rate of rats after growth by downregulating the expression of neuropeptide Y (NPY) and agouti-gene-related protein (AgRP) mRNA and peptide expression in hypothalamic arcuate nucleus (Shi et al., 2009). Evodiamine, a new non-irritant vanillic acid receptor agonist, can simultaneously induce heat loss and heat production, dissipate food energy, and prevent visceral fat accumulation and weight gain (Kobayashi et al., 2001). Then, evodiamine can also activate AMPK and adiponectin polymerization in 3T3-L1 adipocytes, which is related to the activation of Ca²⁺-dependent PI3K/Akt/CaMKII signal pathway (Liu et al., 2014). Treatment with evodiamine over 13 weeks has been shown to lower serum total cholesterol, low and high-density lipoprotein cholesterol, and triglycerides in obese rats on a high-fat diet, decrease blood lipids, and mitigate obesity-related symptoms (Zhang et al., 2017). The combination of berberine and evodiamine can affect the protein expression of PPARγ and liver X receptor α in hyperlipidemic rats and reduce the level of blood cholesterol in hyperlipidemic rats (Zhou et al., 2017b). Furthermore, evodiamine acts as a berberine promoter, and the mechanism of synergistic reduction of serum cholesterol in rats involves inhibiting the expression of Acetyl-CoA Acetyltransferase 2 (ACAT2), Niemann-Pick C1-like 1 (NPC1L1) and apolipoprotein B-48 (apoB-48) to reduce blood lipids (Zhou et al., 2017a). Moreover, ruteacarpine inhibited the expression of NPY and AgRP in the hypothalamic arcuate nucleus and the expression of these two neuropeptides in N29-4 neuronal cells. This method effectively lowers blood cholesterol, non-fasting glucose, insulin, and leptin and ameliorates obesity (Kim S. J. et al., 2009).

On top of that, EF can also contribute to glucose metabolism and improve the symptoms of diabetes. Studies have found that low-dose evodiamine can prevent increased body weight and improve glucose tolerance in mice. Enhanced phosphorylation of AMPK and reduced mTOR signal transduction, a regulator of energy metabolism, are observed in white adipose tissue and are known to avert obesity and insulin resistance (Yamashita et al., 2015). Evodiamide inhibits insulin-stimulated mTOR-S6K activation and IRS1 serine phosphorylation in adipocytes and improves glucose tolerance in obese/diabetes mice (Wang T. et al., 2013). Evodiamine and rutaecarpine can inhibit gluconeogenesis and adipogenesis by activating constitutive androstane receptor (CAR) in vitro and in vivo and have therapeutic potential for the treatment of hyperglycemia and diabetes mellitus type 2 (Yu et al., 2016). Rutaecarpine can regulate the IRS1/PI3K/AKT signal pathway in the liver and AMPK/acetyl-CoA carboxylase 2 signal pathway in skeletal muscle to improve hyperlipidemia and hyperglycemia in fat-fed and streptozotocin-treated rats (Nie et al., 2016). Beyond that, EF polysaccharides extracted by water solvent have strong antioxidant activity and α-glucosidase inhibition. It is a promising natural antioxidant and α-glucosidase inhibitor.

EF can warm the liver and kidney, protect the liver and kidney, and is closely related to evodiamine, rutaecarpine, and limonin. In the liver, evodiamine can promote the translocation of Nrf2 into the nucleus, thereby reducing reactive oxygen species (ROS) levels and oxidative stress in grass carp hepatocytes. It also downregulates the MAPK pathway, alleviating DEHP-induced apoptosis and restoring the expression of antioxidant genes. By blocking the Nrf2/MAPK pathway, evodiamine inhibits DEHP-induced apoptosis in grass carp hepatocytes (Lei et al., 2023; Xiong et al., 2022). Evodiamine (15 and 25 mg/kg) has an anti-fibrotic effect on CCl4-induced hepatic fibrosis and reduces the proliferation and collagen metabolism of hepatic stellate cells in vitro by down-regulating the relative expressions of TGF-β1, p-Smad2/3, and α-smooth muscle actin (Yang D. et al., 2018). Additionally, rutaecarpine upregulates antioxidant enzymes through CaMKII-Akt and Nrf2/antioxidant response element (ARE) pathways, enhances the expression of HO-1 in hepatocytes, and has a protective effect on hepatotoxicity induced by TBHP (Jin et al., 2017). Rutaecarpine protects mice from acute acetaminophen-induced liver injury by activating antioxidant enzymes. It could significantly reduce the activity of serum ALT/AST and MDA induced by acetaminophen and prevent liver GSH depletion induced by acetaminophen (Choi et al., 2021). Moreover, limonins have a furan ring structure and are easily activated to form RMs, which are crucial in induced hepatotoxicity (Liu Y. et al., 2020). CYP3A4 inducer aggravated the hepatotoxicity induced by large flower germ, while limonin reduced its hepatotoxicity (Zhang et al., 2021c). Limonin can also diminish the liver toxicity caused by acetaminophen by activating the Nrf2 antioxidant signal and suppressing NF-κB inflammation by increasing SIRT1 levels. Limonin shows potential as a treatment for liver damage caused by acetaminophen (Yang R. et al., 2020).

In renal metabolism, I/R injury can lead to acute kidney failure. Owing to its antioxidant, anti-inflammatory, and anti-apoptotic properties, evodiamine can reduce the biochemical and pathological tissue of renal I/R injury in rats (Eraslan et al., 2019). Studies have shown that evodiamine can also protect against LPS-induced acute renal injury and cytotoxicity by regulating ROS NF-κB-mediated inflammation (Shi et al., 2019). What is more, rutaecarpine can prevent and treat renal I/R injury by inhibiting JNK/p38 MAPK signal pathway and interfering with oxidative stress (Wang et al., 2017). Moreover, limonin, acting as a natural ERK2 agonist, plays a role in averting ischemic acute renal damage, primarily through the activation of the ERK signal pathway, which aids in the growth of renal tubular cells and diminishes apoptosis following acute kidney injury (AKI) (Zhou et al., 2023). Limonin regulates arachidonic acid metabolism by inhibiting CYP3A4 activity, thus improving cisplatin-induced acute renal injury and ultimately protecting renal function (Zeng et al., 2023).

Ancient records indicate that EF possesses insecticidal properties. The extracts and metabolites of EF showed specific insecticidal properties. Findings indicated superior deworming effects of ethyl acetate, petroleum ether, and methanol extract on Goldfish-Gyrodactylus kobayashii in living organisms, with EF ethyl acetate extract emerging as the most efficient and secure (Lian et al., 2019). Subsequently, EF volatile oil exhibits insect-killing properties against maize weevils, Sitophilus zeamais, and red flour beetle Tribolium castaneum, showing LC₅₀ values of 36.89, 24.57, and 57.31 mg/L air, in that order (Liu and Du, 2011). In addition, evodiamine and rutaecarpine have insecticidal activity against larvae of Drosophila melanogaster with LC₅₀ values of 0.30 and 0.28 μmol/mL diet, respectively, among which rutaecarpine has the strongest activity (Miyazawa et al., 2002). Evodiamine and rutaecarpine had strong insecticidal activity against the fourth instar larvae of Aedes albopictus with LC₅₀ values of 12.51 and 17.02 μg/mL, respectively. EF ethanol extract, limonin, and evodiol also had insecticidal activity against Asian tiger mosquitoes, and the LC₅₀ values were 43.21, 32.43, and 52.22 μg/mL, respectively (Liu et al., 2012). Moreover, the nematicidal activity of evodiamine and rutaecarpine against Meloidogyne incognita was stronger than the crude EF ethanol extract, and the LC₅₀ values were 73.55, 120.85, and 131.54 μg/mL, respectively. Both evodiol and limonin demonstrated their ability to kill Meloidogyne incognita, evidenced by LC₅₀ values of 155.02 and 197.37 μg/mL, respectively, yet they were less potent than the raw EF ethanol extract (Liu et al., 2013). Other studies found that limonin had effective biological activity against larvae and adults of Schistosoma mansoni, and its antiparasite activity was enhanced in a dose-dependent manner (Eraky et al., 2016).

EF’s long-term use in treating diarrhea and beriberi is due to its antibacterial and antifungal properties. The EF volatile oil has the strongest activity against Bacillus subtilis and Staphylococcus aureus, the maximum inhibitory zone diameter is 17.9 and 12.2 mm, respectively, and the MIC is 3.2–6.4 mg/mL (Liu S. S. et al., 2019). Furthermore, three novel quinazoline alkaloids, specifically evodiamine A, evodiamine B, and evodiamine C, were extracted from EF methanol. They prohibited excellent inhibition against Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Xanthomonas campestris pv. campestris, with respective EC₅₀ values of 3.13, 14.32, and 32.72 nmol (Su et al., 2018). Moreover, evodiamine can enhance the activation of NLRP-3 inflammatory bodies by inducing acetylation of α-tubulin lysine 40 residues, thus enhancing innate immunity to bacterial infection (Li C. G. et al., 2019).

Rutaecarpine, evodiamine, and limonin demonstrated obvious anti-osteoporotic effects. Rutaecarpine significantly inhibits osteoclast production and bone resorption of bone marrow-derived macrophages osteoclasts by reducing the protein level of nuclear factor of activated T cells 1 (NFATc-1) and phosphorylation of other signal pathways during osteoclast differentiation (Fukuma et al., 2018). In addition, evodiamine has been reported to inhibit osteoclast formation by blocking receptor activators for NF-κB ligand (RANKL)-induced ERK and c-Fos activation and NFATc-1 induction in a dose and time-dependent manner (Jiang et al., 2017). Evodiamine can inhibit osteoclast formation induced by RANKL through NF-κB and calcium signaling pathways and reduce bone loss in ovariectomy and ovariectomized mice by inhibiting osteoclast production (Jin et al., 2019). Subsequently, evodiamine is capable of mitigating osteoporosis in zebrafish caused by dexamethasone, by counteracting the disproportion in bone development and resorption and triggering the matrix metalloproteinase 3-osteopontin-MAPK pathway signal (Yin et al., 2019). Beyond that, limonin can increase the calcium concentration of the femur and fifth lumbar vertebra in ovariectomized rats, and the mechanism may be related to promoting bone formation (Mandadi et al., 2009). Research has found that the loss of ovarian function can lead to a lack of ovarian-related hormones, resulting in rapid loss of ovarian-related bones (Renno et al., 2006). Limonin can efficiently prevent bone mass reduction and enhance bone mineral density in rats post-ovariectomy. Moreover, limonin stimulates the activity of ALP in osteoblast MC3T3-E1 and enhances the expression of osteoblast differentiation gene markers by regulating extracellular signal-regulated kinase and P38 signal (Lee et al., 2016).

EF not only offers attractive pharmacological effects but also provides antiallergic, antioxidant, antidepressant, and protection for the prostate, among others.

EF causes hepatocyte cytotoxicity, attributed to oxidative stress, mitochondrial damage, endoplasmic reticulum stress, liver metabolic disorder, and apoptosis (Cai et al., 2014). The toxicological mechanisms are peroxidation, inflammatory factors, mitochondrial damage, and the formation of drug-protein adducts. Research revealed that various EF extracts might lead to sudden liver damage, with volatile oil exhibiting the highest level of hepatotoxicity, succeeded by total extract and ethanol extract, and water extract showing minimal hepatotoxicity.

The cardiotoxicity of EF is mainly caused by oxidative damage and inhibition of human ether-a-go-go-related gene (hERG) channels in the heart. Its primary connections are in alkaloids with evodiamine, rutaecarpine, dehydroevodiamine, and hydroxyrutaecarpine.

EF’s nephrotoxic effects primarily stem from renal cell death and oxidative stress. Its similarity to evodiamine and limonin in EF is notable. Mice were administered the EF ethanol extract in groups of low, medium, and high dosages. In the group receiving a high dosage, there was a noticeable flexing of the glomerular Mesangium and an enlargement of both glomerular podocytes and endothelial cells (Liu et al., 2015). In vivo experiments showed that evodiamine could induce renal cell death and regulate the PI3K/AKT/mTOR pathway by inducing intracellular calcium overload (Yang et al., 2024a). Rutaecarpine can significantly reduce the level of cortisol and regulate glucocorticoid metabolism. Renal injury may be related to the induction of apoptosis-related protein Bax and Bcl2 expression (Yang C. Q. et al., 2022). Evodiamine and evodiolide in alkaloids can damage mitochondria, lead to mitochondrial dysfunction, produce a large number of free radicals from mitochondria, further aggravate oxidative stress, induce apoptosis, and promote renal damage (Zhou Q.j. et al., 2013). In addition, limonin also has a certain toxicity to kidney cells. Comprehensive analysis of animal experiments and chromatographic analysis showed that hydroxyl or acetoxy limonoid derivatives and coumarin in EF may be the leading causes of toxicity (Shan et al., 2021). Studies have shown that limonin (50–200 μg/mL) can significantly inhibit the viability of HEK 293 cells in a dose-dependent manner. A concentration ranging from 100–200 μg/mL may lead to atrophy, reduction, or even fatality of renal cells (Fan et al., 2019).

The typical oral dose of EF is 2–5 g, taken by water decoction or pill powder. It is generally non-toxic within a reasonable dose range, but it needs to be monitored if it is overused. The liver function test, coagulation function test, serum bilirubin level test, liver ultrasound, and liver biopsy can monitor the hepatotoxicity of EF. Liver function tests can detect transaminase, bilirubin, alkaline thrombin, cholinesterase, and other indexes in blood and evaluate the functional status of the liver (Shiihara et al., 2024). Coagulation function tests can determine prothrombin time, thrombin time, and activated thrombin time and help to judge whether the liver is abnormal (Kumagai et al., 2023). A high serum bilirubin level may indicate liver injury or biliary obstruction disease (Poynard et al., 2023). Liver ultrasound can observe liver structural abnormalities or steatosis (Vardar et al., 2022). Liver biopsies help to confirm whether there is hepatocyte injury or inflammation (Palmer et al., 2024). Therefore, patients using EF should regularly check their liver function and blood coagulation function, pay close attention to the indexes of transaminase, bilirubin, alkaline thrombin, and cholinesterase, and adjust the dosage of EF in time to avoid drug-induced liver injury.

The cardiotoxicity of EF can be evaluated by physical monitoring and measurement of specific biomarkers, including electrocardiogram changes, blood electrolyte levels, and myocardial enzyme activity (Qiu et al., 2023). An electrocardiogram is a direct indicator of the heart’s electrophysiological function, and irregular waveforms could signal the presence of an arrhythmia (Henriksen et al., 2023). Disproportionate levels of electrolytes in the blood, particularly irregular potassium ion concentrations, frequently lead to arrhythmias (Sauer and Porter, 2021). Increased myocardial enzyme activity, such as creatine kinase and lactate dehydrogenase, may damage heart tissue (Gai et al., 2021). Therefore, patients using EF should have regular ambulatory electrocardiogram monitoring. The global cardiac activity was measured for 24 h, and the heart rate variability and myocardial perfusion indexes were observed. It assists patients in adjusting their medication dosage promptly to reduce the occurrence of cardiotoxicity.

The nephrotoxicity of EF can be monitored by urinalysis, renal function tests, biochemical assays, and renal biopsy (Canki et al., 2024). Urine tests can reflect proteinuria, hematuria, hemoglobinuria, cylindruria, urinary calcium, and alkaline urine (Wu and Fenton, 2018). Renal function tests assess the endogenous creatinine clearance rate, blood urea nitrogen, creatinine, and uric acid levels and evaluate the glomerular filtration rate through a radionuclide renogram (Nieto et al., 2020). Biochemical tests can monitor serum liver and renal function-related enzyme levels (Sumer et al., 2020). A renal biopsy is capable of directly monitoring the pathological alterations in kidney tissue, including the deterioration of tubular epithelial cells, necrosis, congestion in the interstitial space, swelling, infiltration of inflammatory cells, fibrosis in the renal interstitial area, and the expansion or shrinkage of the renal tubules (Xu et al., 2024). Therefore, patients taking EF should have regular urinalysis and blood tests and pay attention to the levels of urinary protein, red blood cells, serum creatinine, and urea nitrogen to ensure timely detection of issues and implementation of appropriate treatment measures, thereby reducing the incidence of renal toxicity. Concurrently, it is crucial to rigorously regulate the dosage while administering EF to prevent excessive use. Regular monitoring of kidney function during usage is crucial for early identification of kidney toxicity.