Traditional Chinese medicine

What is Traditional Chinese Medicine? Traditional Chinese Medicine (TCM) is a complete system of medicine developed to prevent and treat disease. Its roots are in writings and practices which were developed over millennia. TCM uses acupuncture, herbs and other natural substances, acupressure, food therapy, meditation and exercise to treat disease and maintain health. The Chinese medical view is that each living creature is a small part of the infinite universe, and is subject to the same laws that govern the rest of the cosmos. Therefore, an understanding of health requires an understanding of the laws of nature. The practitioner views health as a state of harmony existing between the internal environment of the body and the external environment it lives in. Unfavorable climactic conditions, emotional upset, physical trauma, infectious organisms, poor nutrition, inappropriate lifestyle, heredity, and other pathogenic factors are capable of disrupting this state of harmony. TCM developed a metaphoric or schematic model for how the internal body operates that incorporated the same principles of balance and harmony that govern the ecology of the natural world. Health and disease are viewed simply as two different self-perpetuating cycles, both of which are resistant to change. Organ interactions are the stepping stones in each cycle. When they are mutually supportive, the spinning of the cycle allows the organism to throw off illness. When they are mutually antagonistic, the spinning of the cycle makes the organism resistant to all but therapieswhich directly address the stepping stones in the cycle of disease. From the TCM perspective, chronic disease is not chaos, but a highly stable state, which explains why it is so difficult to resolve. Most medical systems in the world except modern medicine lacked definitive knowledge about the inner workings of the body due largely to cultural taboos. In order to address illness in the absence of detailed medical knowledge, most cultures developed schematic or metaphoric models for how the body worked. Almost all of these systems believed that individuals have inherent strengths and weaknesses. Since we are born with these weaknesses, they are viewed to pervade our systems and to have somehow contributed to most of our health problems, even if they appear to result in unrelated diagnoses from a conventional perspective. These weaknesses are echoed in everything, including our behaviors, our appetites, our childhood illnesses, and even how our tongue looks or our pulse feels. Chinese medical diagnosticians take a …


Introduction
Euodiae Fructus (EF), known as "Wuzhuyu" in China, "Goshuyu" in Japan and "Osuyu" in Korea, are the dried and nearly ripe fruits of Euodia rutaecarpa (Juss.) Benth. (ER), E. rutaecarpa (Juss.) Benth var. officinalis (Dode) Huang (ERO), and E. rutaecarpa (Juss.) Benth. var. bodinieri (Dode) Huang (ERB). It has been used as traditional Chinese medicine (TCM) for more than 2000 years and is officially listed in multiple versions of Chinese Pharmacopoeia. At the same time, it is also traditionally and ethnically used in Japan and Korea. According to the records of TCM, Euodiae Fructus could be widely used either alone or in combination with other herbal medicines as remedies for gastrointestinal disorders (abdominal pain, dysentery), headache, emesis, aphtha, dermatophytosis, dysentery, amenorrhoea, menorrhalgia and postpartum haemorrhage. However, it is worth noting that irrational use of this herb could cause toxic symptoms such as stomach ache, vomiting, blurred vision, etc.
With the increasing interest paid to the pharmacologically phytochemicals from the Euodiae Fructus, a lot of investigations related to the phytochemical and pharmacological aspects of this plant have been carried out. To date, a variety of chemical constituents, including alkaloids, terpenoids and steroids, as well as phenols and volatile oils, have been isolated and identified from Euodiae Fructus. Pharmacological studies revealed that the crude extracts and purified compound possess a wide spectrum of biological activities, involving in Page 2 of 43 Xiao et al. Natural Products and Bioprospecting (2023) 13:6 anticancer, antibacterial, anti-inflammatory, insecticide, anti-cardiovascular, neuroprotective, anti-obesity and anti-diabetic activities, confirmed by various in vivo and in vitro experiments, as shown in Fig. 1. In recent years, several reviews have been published on the chemical and biological activities of ivodimine [1,2], erythrartine [3,4] and citrinin [5]. A review of Euodiae Fructus is essential for present and future study toward improving phytochemical and pharmacological investigation. Herein, we systematically described and summarized the study advances of Euodiae Fructus in recent decades, including phytochemical, pharmacological effects, toxicity, and quality control. We reviewed the literature up to February 2021.

Botanical descriptions
In Chinese Pharmacopoeia, the dried and nearly ripe fruits of three plants of the genus Euodia rutaecarpa The common botanical characteristics of the above three plants are small trees or shrubs, 3-5 m high. They often have opposite odd-pinnate leaves. Their inflorescences are terminal; the flowers of the male inflorescence are separated from each other, and the flowers of the female inflorescence are dense or separated. The dioecious flowers have 4 or 5 sepals, petals, stamens, and carpels. The fruits are oblate and split into 5 petals when mature, and they are follicle-like, purplish red, with large oil glands on the surface, and each fruit has one seed, black and shiny. The flowering period ranges from June to August, and the fruit period is typically from August to November. However, there are also some differences of them in botanical descriptions and distribution area, as shown in Table 1.
These three plants usually grow in mountains, roadsides, or sparse forests. It is mainly produced in the southern regions of China (such as Hunan, Guizhou, Sichuan, Yunnan), as well as in Japan, Korea, Bhutan, northeast India, Myanmar, and Nepal.

Traditional uses
Euodiae Fructus has a long history as a traditional remedy and has been widely used Chinese medicine as recorded in the ancient herbal books and Pharmacopoeia of the People's Republic of China (Editorial Committee of Chinese Pharmacopoeia, 2020). According to the history of TCM, Euodiae Fructus, initially recorded in "Shen Nong's Herbal Classic", is listed as a middle-grade herbal item and also described as being pungent and bitter in taste and can return to the liver, spleen, stomach and kidney meridians. According to the records of "Ri Hua Zi Ben Cao", it could strengthen the spleen, treat abdominal pain, beriberi, edema, and postpartum haemorrhage. Furthermore, it was found to kill harmful insects and prevent tooth decay in "Ben Cao Shi Yi". As it was recorded in "Compendium of Materia Medica", the main function of Euodiae Fructus was to improve digestion, relieve headache, abdominal pain and treat hemorrhoids in throat, mouth and tongue [6]. According to the 2020 Edition of Chinese Pharmacopoeia, Euodiae Fructus is often used for external use and the recommended dosage is 2-5 g, the "standard" processing method of Euodiae Fructus is stir-frying with licorice water extract, other usual processing methods include washing with hot or cold water [7]. Since the compatibility of medicines is considered to improve effects, reduce toxicity, or achieve synergistic or balanced effects [7]. Euodiae Fructus was often combined with Jujubae Fructus, which has the effect of treating stomachache and pregnancy headache. If combined with Angelicae Sinensis Radix, it could promote blood circulation and relieve menstrual pain. When combined with Zingiberis Rhizoma Recens, it could promote yang and dispel cold. Moreover, Euodiae Fructus could be used in a combination with Codonopsis Radix or Foeniculi Fructus, thereby playing a significant role in tonifying and warming stomach, etc. Based on the above compatibility, Euodiae Fructus was typically used in polyherbal formulations in TCM (http:// www. zysj. com. cn/ zhong yaofa ng/ index. html), and the composition and therapeutic effects of typical polyherbal formulations are summarized in Table 2. In recent years, numerous studies in vitro and vivo have indicated that "Zuo Jin Wan" (ZJW) possess good pharmacological effects, such as anti-inflammation, anti-ulcer [8], anti-acid [9], antidepressant-like [10], and anti-cancer properties [11]. Noteworthy, Li et al.
conducted a systematic review and meta-analysis according to a total of 1736 patients in 18 studies, indicating "Wenjing Tang" was shown to be significantly superior to nonsteroidal anti-inflammatory drugs in improving primary dysmenorrhea in terms of clinical effective rate, the visual analogue scale, and the pain scale for dysmenorrhea [12].
Besides, Euodiae Fructus is also popular in Japan and South Korea. According to Dongui Bogam, a representative Korean Medicine book, Euodiae Fructus has been frequently used as a prescription for treating headache, abdominal pain, vomiting, cold, reducing blood circulation and gynecological diseases (amenorrhea), with a dose of 2-8 g. It is also one of the main components of traditional herbal prescriptions for the treatment of sterility caused by irregular menstruation such as Chokyungjongok-Tang, Nangungjongsa-whan, and Onkyung-Tang [13]. In addition, Euodiae Fructus was introduced in Japan as early as Edo, mainly applied for the treatment of cold and pain. For example, Goshuyuto, a representative traditional Japanese medicine, also known as "Wuzhuyu Tang" in China and "Osuyu-tang" in Korea, is composed of four medicinal herbs, Euodiae Fructus, Ginseng Radix Et Rhizoma, Zingiberis Rhizoma Recens, Jujubae Fructus, and it could be used to treat migraine headache, nausea, beriberi, and heart failure [14].

Phytochemistry
To date, more than 240 kinds of constituents have been isolated and identified from Euodiae Fructus, including 133 alkaloids, 36 terpenoids, 5 steroids, 51 phenols and 15 other compounds. Among them, alkaloids and terpenoids have been identified as the characteristic components. All compounds are summarized and compiled in Table 3. Table 1 The differences in botanical descriptions between three plants

Plant Botanical description differences Distribution area
Euodia rutaecarpa (Juss.) Benth Its leaflets are larger, up to 7 cm wide, and slightly thicker than paper, and both sides are densely hairy; its rachis of the inflorescence are reddish-brown hairy, and the female inflorescence has denser flowers, with petals up to 5 mm long and densely hairy on the inner surface.; its fruit stalk is shorter and stout It is mainly distributed in Guangdong, Guangxi              188 191

Alkaloids
The alkaloids extracted from Euodiae Fructus have attracted wide attention from chemists and pharmacologists due to their various biological effects. Among these compounds, indole alkaloids and quinolone alkaloids are the main structural types. Up to 53 indole alkaloids were isolated from Euodiae Fructus, and their structures are shown in Fig. 2. Evodiamine, rutaecarpine and dehydroevodiamine are regarded as the dominant chemical constituents with a wide range of pharmacological activities.
Moreover, several phenolic acids and their derivatives have also been found in Euodiae Fructus. In 2013, caffeic acid (208) was isolated from the genus Evodia for the first time [81]. In recent years, He et al.  (204) [82], and isovanillin (223), were successfully extracted from Euodiae Fructus.

Volatile oil
The volatile oil is one of the main chemical compositions of Euodiae Fructus and its content is very high. Liu et al. identified 97 constituents by gas chromatography/ mass spectrometer (GC/MS) analysis from 24 samples [91]. Another study showed that 97 constituents identified by SPME-GC-MS, accounted for 96.80% of volatile oil. Among the isolated volatile oil, the relative content of sesquiterpenes was more than 38%, monoterpenoids components was over 35%, ester components were above 13% [92]. It also indicated that the main constituents of the volatile oil from Euodiae Fructus were β-myrcene (17.7%), (Z)-β-ocimene (14.8%), α-phellandrene (14.7%), γ-terpinene (6.4%), linalool (5.7%) and β-thujene (5.1%) [93]. Moreover, several researches have been reported the volatile constituents obtained from Euodiae Fructus, such as caryophyllene oxide, linalool and γ-Elemene, have diverse functions, such as sedative, antiasthmatic, antibacterial, antitumor, antiviral and insect repellent, and its main components are caryophyllene oxide. It has been found that elemene is a new anticancer drug with great potential and has a broad clinical application prospect. Meanwhile, γ-Elemene can promote the immune function of erythrocytes [94]. However, modern toxicology studies showed that volatile oil could induce certain acute liver damage [95]. Taken together, the volatile oil may be efficacy material basis and toxicity material basis, but the research is isolated and lack of correlation, so further studies need be conducted to provide the experimental data and literature evidence for reasonable and safe development of the volatile oil from Evodia Fructus.

Pharmacology
As a well-known medicinal plant in TCM, Euodiae Fructus has been validated to possess a diverse set of pharmacological properties, such as anticancer activity [96,97], antibacterial activity [98], anti-inflammatory activity [45,99,100], insecticide activity [31], antinociceptive activity [101], and anti-diarrheal effect [102]. Evodiamine [1], rutaecarpine [4], and limonin [5], which are major compounds of this plant and display a variety of biochemical and pharmacological properties in the cancer, cardiovascular, central nervous system and so on, and it is possible to be developed as a promising lead compound for drug discovery. All the detailed information is shown in Table 3.

Anticancer activity
In crude extracts, the methanol extract of Euodiae Fructus decreased the AP-1 stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 transactivation in Chang/AP-1 cells, with an EC 50 value of 24.72 μg/ mL [103]. Park et al. found that the 70% ethanol extract of Euodiae Fructus could induce apoptosis in HeLa cells with IC 50 of about 45 μg/mL, which may be associated with a caspase-dependent cascade through activation of the intrinsic signaling pathway connected with AMPactivated protein kinase activation [96]. Another study showed that the 70% ethanol extract of Euodiae Fructus could inhibit the growth of benign prostatic hyperplasia-1 (BPH-1) epithelial cells by inhibiting proteins and antigens including 5α-reductase, proliferating cell nuclear antigen (PCNA), phosphor-ERK1/2, and cyclin D1 and by inhibiting cell viability dependently through the activation of caspase-3 and caspase-8 [97]. Additionally, ZJP aqueous extract exhibited its prominent therapeutic effects on hepatocellular carcinoma (HCC) mainly via the regulation of cell proliferation and survival though the EGFR/MAPK, PI3K/NF-κB, and CCND1 signaling pathways [104].
Numerous in vitro studies have reported that the isolated compounds of Euodiae Fructus display antitumor activities in several cancer cell lines, and the detailed information is presented in Table 3 Table 4.

Antibacterial and antifungal activity
Euodiae Fructus has been used to treat infection-related diseases including diarrhea, beriberi and oral ulcer for a long time due to its antibacterial and antifungal activities.  [91].
In isolated compounds, the two novel alkyl methyl quinolone alkaloids (compounds 80-81) (AM quinolones) shown highly selective antimicrobial activity against H. pylori without harmful adverse effects against other intestinal flora [56], thereby being a candidate for use in eradication therapy for H. pylori in vitro and vivo [217]. In addition, evodiamine was able to augment the NLRP3 inflammasome activation by inducing acetylation at K40 residue of α-tubulin, thus conferring intensified innate immunity against bacterial infection [197].

Anti-inflammatory and analgesic activity
Euodiae Fructus has been used in TCM for the treatment of inflammation-related disorders such as gastrointestinal disorders (gastric ulceration, ulcerative colitis and dysentery), headache, postpartum hemorrhage, amenorrhea and dermatitis [121]. Numerous studies have demonstrated that dysregulation of nuclear factor-kappa B (NF-кB) pathways and inflammatory factors, such as TNF-α, IL-1β, IL-6 and NO, etc. play important roles in inflammatory responses [218].

Anti-inflammatory activity
The water extract of Euodiae Fructus could enhance the gastric mucosal barrier and promote the synthesis of NO in gastric mucosa, which has a significant protective effect toward ethanol-induced gastric injury in rats [219]. Ko et al. showed that the ethanol extract of Euodiae Fructus display potent antioxidative effects against both phorbol-12-myristate-13-acetate (PMA)-and N-formyl-methionyl-leucyl-phenylalanine (fMLP)induced ROS production in neutrophils with respective IC 50 values of 2.7 and 3.3 μg/mL and also inhibit lipopolysaccharide (LPS)-induced NO production with an IC 50 of around 0.8 μg/mL, suggesting that the ethanol extract exhibited anti-inflammatory activities which could be partially explained by inhibiting NADPH oxidase-dependent ROS and/or iNOS-dependent NO production in activated inflammatory cells [118]. In another study, Euodiae Fructus and its active components may be useful in influenza virus infection-related inflammatory disorders by suppressing novel influenza A (H1N1)induced chemokines (RANTES and MCP-1) production and blocking chemokine-attracted leukocytes recruitment [100].
In isolated compounds, results have showed that the anti-inflammatory effect of rutaecarpine is partly ascribed to the diminution of prostaglandin (PG) production through inhibition of arachidonic acid release in the RAW 264.7 [119]. In other studies, rutaecarpine ameliorated sepsis-induced peritoneal resident macrophages apoptosis and inflammation responses through inhibition of endoplasmic reticulum stress-mediated caspase-12 and NF-κB pathways [120], improved imiquimod-induced psoriasis-like dermatitis through effects on pDC-and Th17-associated cytokines via modulation of NF-κB and toll-like receptor 7 (TLR7) signaling [220], and ameliorated dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) via inhibiting KEAP1-NRF2 interaction to activate NRF2 [122]. Similarly, limonin was reported to improve the prognosis of DSS-induced UC mainly through downregulating p-STAT3/miR-214 levels    [128]. Moreover, evodiamine could improve antioxidant and anti-inflammatory status through Rho/NF-κB pathway, which possibly exerted a gastro-protective effect against gastric ulceration [123]. In vitro and vivo, evodiamine was able to protect against zymosan-induced inflammation and DSS-induced murine experimental colitis by inactivating the expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), NF-κB signal pathway [124] and NLRP3 inflammasome [221], and the antiarthritic effect of evodiamine might be associated with its repression of synovial inflammation and regulation of Treg and Th17 differentiation [125].

Antinociceptive activity
Moreover, it has been reported that oral administration of 50 or 200 mg/kg 70% methanol extract of Euodiae Fructus has an antinociceptive effect on acetic acid induced-writhing responses, and the mode of action may be mediated by its anti-inflammatory action [101]. In vivo, limonin (30 or 100 mg/kg) possessed an antinociceptive effect and the effect may be accompanied by an anti-inflammatory action [69]. In other studies, evodiamine could reduce capsaicin-induced currents significantly in vitro and suppress capsaicin-induced thermal hyperalgesia in rats, which may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons [212], and it could also inhibit the migraine-like pain response possibly due to the regulation of nNOS and suppression of the AMPA receptor GluA1 [126].

Anti-cardiovascular disease activity
Several studies have demonstrated that Euodiae Fructus has anti-cardiovascular activities, such as vasoconstrictive and vasodilator effects, anti-atherosclerosis, anti-platelet aggregation, anti-thrombus, anti-arrhythmia and cardioprotective effects [3].

Vasoconstrictive and vasodilator activity
It has been found that Euodiae Fructus (1 × 10 −6 -3 × 10 -4 g/mL) has constrictive effects on rat aorta via adrenergic α 1 receptors and serotonergic (5-HT 1D and 5-HT 2A ) receptors [65], and the effect toward calcium channel on the membrane also played important roles [222]. In other investigations, rutaecarpine produced a fully (100%) NO-dependent vasodilatation in rat aorta, whereas dehydroevodiamine and evodiamine produced a partially endothelium-dependent effect, 10% and 50%, respectively. Furthermore, multiple-action mechanisms, including endothelium dependence, α 1 -adrenoceptor blockade, K + channel activation, and Ca 2+ channel blockade were probably involved in the vasorelaxant effects of dehydroevodiamine [223]. In vivo and vitro, the depressor and vasodilator effects of rutaecarpine were related to stimulation of endogenous CGRP release via activation of vanilloid receptors [181,224].

Modulatory effects on VSMCs function and intimal hyperplasia
Results showed that evodiamine suppressed oxidative stress and inflammatory responses due to high free fatty acids and high glucose in human umbilical vein endothelial cells (HUVECs) via inhibiting the upregulated expression of P2X4R signaling pathway [179] and P2X 7 receptor [180], respectively. Further investigations have shown that a promising anti-atherogenic effect of evodiamine through attenuation of vascular smooth muscle cells (VSMCs) migration by suppressing cell cycle progression, p38 MAPK and Erk1/2 activation, and ROS generation [178], and the activation of PPARγ also plays important role [225]. It was worth noting that rutaecarpine could modulate Cx (theroprotective Cx37 and atherogenic Cx43) expression through TRPV1/[Ca 2+ ] i/CaM/NF-κB signal pathway [174] in monocytes to enhance its antiadhesive properties [171,175], thereby preventing VSMCs dysfunction induced by ox-LDL [176]. Additionally, rutaecarpine inhibited Angiotensin II-induced proliferation in VSMCs partly through the modulation of NO signaling pathways and other related molecules (HRG-1 and c-myc) [173]. Moreover, rutaecarpine (10, 20, and 40 mg/kg) suppressed atherosclerosis in ApoE −/− mice through upregulating ABCA1 and SR-BI within reverse cholesterol transport (RCT) [177], and it could also promote NO production and inhibit ERK2 signal transduction pathways to inhibit the balloon injuryinduced carotid intimal hyperplasia in rats [183].

Anti-platelet activity
"Goshuyuto" at the concentration of 1000 μg/mL inhibited collagen-induced platelet hyper-aggregation to the same degree as aspirin at the concentration of 100 μM [166]. Rutaecarpine was also able to display an antiplatelet effect in vivo [167], and the mechanism was investigated by inhibition of thromboxane formation and phosphoinositide breakdown [168]. Further investigation has shown that rutaecarpine inhibits agonists-induced platelet aggregation in human platelets, probably by inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by inhibition of thromboxane A 2 formation and [Ca 2+ ] i mobilization [169]. In another study, rutaecarpine has been seen to exert both antihypertensive and anti-platelet effects by stimulating the synthesis and release of CGRP in spontaneously hypertensive rats (SHR), and CGRP-mediated     Evodiamine In vivo Male SD rats with myocardial I/R injury 30 or 60 μg/kg [188] Evodiamine In vitro TGF-β1-induced neonatal rat cardiac fibroblasts 1, 5, and 10 μM [189] Evodiamine In vitro Angiotensin II-induced rat cardiomyocyte hypertrophy 0.3, 3 μM [190] Evodiamine In vivo Male C57BL/6 mice with isoproterenol-induced cardiac fibrosis 50 and 100 mg/kg [191] Anti-obesity and anti-diabetic activity

Anti-arrhythmia activity
It has also been found that evodiamine and rutaecarpine induce the positive inotropic and chronotropic effects on the guinea-pig isolated right atria through their interaction with vanilloid receptors and the resultant release of CGRP [226,227]. Additionally, dehydroevodiamine (0.1-0.3 μM) could depress trigger arrhythmias in Caoverloaded guinea-pig cardiac myocytes through inhibiting I Na , I ti and, to a smaller extent, I Ca , while increasing the intracellular pH (pH i ) and Na + -H + exchanger (NHE) activity [228].

Regulatory effects on cardiac injury
Yi et al. found that the protective effects of rutaecarpine on cardiac anaphylactic injury or ischemia-reperfusion injury were related to inhibition of TNF-α production by stimulation of CGRP release [184], and the involvement of capsaicin-sensitive sensory nerves also played important roles [185], and the inhibition of Nox4-ROS-ADAM17 pathway and over-activation of ERK1/2 might be associated with the beneficial role of rutaecarpine in hypertensive cardiac hypertrophy [182]. Moreover, evodiamine (0.3 and 3 μM) significantly attenuated Ang II-induced cardiomyocyte hypertrophy in vitro, and this effect is partly due to the promotion of NO production, the reduction of [Ca 2+ ] i concentration, and the inhibition of CaN and ERK-2 signal transduction pathways [190], and it could also prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against endothelial to mesenchymal transition (EndMT) probably by inhibition of canonical [189] and non-canonical TGFβ signaling [191].

Neuroprotective activity
A wide spectrum of pharmacological experiments indicated that Euodiae Fructus and its isolated compounds exerted a neuroprotective effect against ischemic injury, neuropathic pain, nerve inflammation, neurodegenerative disorders such as Alzheimer's disease (AD), etc. The methanol extract of Euodiae Fructus (200 mg/kg) was able to have a protective effect against ischemiainduced neuronal and cognitive impairment [114]. In a MDCK-pHaMDR cell monolayer model, evodiamine and rutaecarpine entered the blood-brain barrier (BBB) by passive diffusion and promoted the absorption of dehydroevodiamine probably by inhibiting P-gp, while dehydroevodiamine showed moderate permeability through Evodiamine In vitro The NRK-52E rat proximal tubular cell line 10 and 20 mg/kg [207] In vivo Male SD rats with lipopolysaccharide-LPSinduced acute kidney injury

Rutaecarpine
In vitro HepG2 cells with t-BHP-induced hepatotoxicity 1-10 μM [208] In vivo Male ICR mice with t-BHP-induced hepatotoxicity BBB by P-gp mediated efflux. Moreover, the above three alkaloids have been confirmed to exhibit neuroprotective effects on MPP + or H 2 O 2 -injured PC12 cells [115]. In other studies, evodiamine (10 μM) and rutaecarpine (50 μM) reduced peripheral hypersensitivity and anxiety in mice with nerve injury or inflammation via TRPV1 [116]. Moreover, evodiamine could ameliorate paclitaxel-induced neuropathic pain by inhibiting inflammatory response and activating mitochondrial anti-oxidant functions [15], and induced JNK-mediated protective autophagy in astrocytes through TRPV1-dependent signaling and an influx of extracellular calcium, which may provide a possible option for ischemic stroke treatment [229]. Additionally, rutaecarpine improved neuronal injury, inhibited apoptosis, inflammation and oxidative stress in rats with cerebral ischemia-reperfusion (CI/R) by regulating the expression of ERK1/2 and Nrf2/HO-1 pathway [117].
Besides the above functions on the nervous system, Euodiae Fructus and its isolated compounds could also potentially be developed as an alternative therapeutic agent for the management of AD. Cai et al. demonstrated that the water extract of Euodiae Fructus significantly ameliorated learning and memory deficits in Morris water maze tests, and in 3xTg AD mice, it could also decrease Aβ deposits and increase NeuN-positive cells by upregulating the expressions of Brain neurotrophic derived factor (BDNF) and tyrosine kinase B (TrkB) [105]. Evodiamine (100 mg/kg) significantly alleviated the impairments of learning ability and memory in transgenic mouse models [112], and inhibited glial cell activation and neuroinflammation (IL-1β, IL-6, TNF-α, and COX-2 levels) in the hippocampus by increasing the activity of AKT/GSK-3β signaling pathway and inhibiting the activity of NF-κB [111]. Further study has revealed that evodiamine exerts a protective effect against AD by modulating oxidative stress and reducing the apoptosis rate in vitro and vivo [113]. Additionally, dehydroevodiamine could inhibit acetylcholinesterase activity with IC 50 value of 37.8 μM and show antiamnesic effect due to the combined effects of acetylcholinesterase inhibition and the known cerebral blood flow enhancement [107], and it could also suppress WT/GFX-induced overactivation of GSK-3 to improve spatial memory impairment and tau hyperphosphorylation in vivo [109], and its underlying mechanism might involve a decreased inhibitory phosphorylation of PP-2A at Tyr307 [108], and the protective effects on cognitive impairment might be related to its antioxidant activity, inhibition of neurotoxicity and intracellular calcium in memory-impaired rat models [110].

Anti-obesity activity
It has been reported that ruteacarpine and evodiamine [193] reduce food intake and bodyweight gain by improving orexigenic sensitivity through the inhibition of neuropeptide Y (NPY) and agouti-related protein (AgRP) mRNA expression and peptide expression [230]. Moreover, evodiamine, as a vanilloid receptor agonist, could simultaneously induce heat loss and heat production and dissipate food energy, preventing the accumulation of perivisceral fat and the body weight increase [231], and activate AMP-activated protein kinase (AMPK) and adiponectin multimerization in 3T3-L1 adipocytes, which was associated with the activation of Ca 2+ -dependent PI3K/Akt/ CaMKII-signaling pathway [192].

Anti-diabetic activity
Furthermore, rutaecarpine and evodiamine were able to suppress gluconeogenesis and lipogenesis through their activation of the constitutive androstane receptor (CAR) in vitro and vivo, thus having a therapeutic potential for the treatment of hyperglycemia and type 2 diabetes [195]. Evodiamine improved glucose tolerance and reduced insulin resistance in obese/diabetic mice, which was possibly related to inhibition of mammalian target of rapamycin (mTOR)-S6 protein kinase (S6K) signaling and insulin receptor substrate 1 (IRS1) serine phosphorylation in adipocytes [194]. An additional study demonstrated that rutaecarpine could regulate IRS-1/PI3K/Akt signaling pathway in liver and AMPK/ acetyl-CoA car-boxylase2 (ACC2) signaling pathway in skeletal muscles to ameliorate hyperlipidemia and hyperglycemia in fatfed, streptozotocin-treated rats [196].

Insecticidal activity
In recent years, plant-based, environmentally friendly and biodegradable natural insecticides have received renewed attention as vector control agents, and some research have demonstrated that Euodiae Fructus exhibit insecticidal activity [232]. Lian et al. screened different extracts of Euodiae Fructus with anthelmintic activity against Gyrodactylus kobayashii (Monogenea) in goldfish, indicating that the ethyl acetate, the petroleum ether and methanol extracts had better anthelmintic efficacy, with EC 50 values of 24.0, 71.9 and 40.9 mg/L, respectively, after a 48-h exposure, whereas the water extract of Euodiae Fructus had the weakest anthelmintic efficacy of 25.6% at 800.0 mg/L [198]. Moreover, the essential oil of Euodiae Fructus was found to possess insecticidal activity against maize weevils, Sitophilus zeamais and red flour beetles Tribolium castaneum with LC 50 values of 36.89, 24.57 and 57.31 mg/L air, respectively [93]. Further study has shown that evodiamine and rutaecarpine showed insecticidal activity against larvae of D melanogaster with LC 50 values of 0.30 and 0.28 μmol/mL diet respectively [199]. In another investigations, evodiamine, rutaecarpine, and wuchuyuamide I have been reported to exhibit strong larvicidal activity against the early fourth instar larvae of A. albopictus with LC 50 [29]. Taken together, these results indicated that the ethanol extract of Euodiae Fructus and several isolated constituents have a good potential as a source for insecticidal activity, and further research is needed to determine its safety to human body and environment.

Hepatorenal protection
Consistent with traditional applications, Euodiae Fructus was reported to affect the liver and kidney [233]. Jin et al. reported that rutaecarpine augmented cellular antioxidant defense capacities through CaMKII-PI3K/ Akt-dependent HO-1 induction via the Nrf2/ARE signaling pathway, thereby protecting cells from oxidative damage in hepatocytes [208]. It has been found that evodiamine (15 and 25 mg/kg) has an antifibrosis effect in CCl 4 -induced liver fibrosis and reduces hepatic stellate cells (HSCs) proliferation and collagen metabolism in vitro through downregulation of relative expression of TGF-β1, p-Smad 2/3, and α-SMA [205]. In other investigation, limonin alleviated acetaminophen-induced hepatotoxicity by activating Nrf2 antioxidative signals and inhibiting NF-κB inflammatory response via upregulating Sirt1 [210]. For the kidney, recent researches showed that a number of protective roles against I/R damage [206], LPS-induced acute kidney injury and cytotoxicity [207] due to the antioxidative, anti-inflammatory and antiapoptotic properties of evodiamine. Additionally, Wang et al. showed that rutaecarpine be an effective compound for the prevention and treatment of renal ischemia-reperfusion injury (IRI), and its mechanism might be related to inhibition of JNK/p38MAPK signaling pathway and interference of oxidative stress response [209].

Anti-osteoporosis activity
Rutaecarpine significantly inhibited osteoclastogenesis and prevented bone resorption of bone marrow-derived macrophage (BMM)-derived osteoclasts through decreasing the protein level of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) and the phosphorylation of other signaling pathways during the osteoclast differentiation [203]. Moreover, evodiamine was reported to inhibit the formation of osteoclasts via blocking the RANKL-induced activation of ERK and c-Fos as well as the induction of NFATc1 [200], and the underlying mechanism might also be related to inhibit the activation of the NF-κB and calcium signalling pathways [201], and in Zebrafish, evodiamine was found to prevent osteoporosis by reversing the imbalance of bone formation/bone resorption and activating MMP3-OPN-MAPK pathway signal [202]. Additionally, limonin stimulated alkaline phosphatase (ALP) activity and enhanced the expression of osteoblast differentiation gene markers by regulating ERK and P38 signals in osteoblastic MC3T3-E1 cells, and inhibited the reduction of bone mass and promote the increase of bone mineral density in ovariectomized rats [204].

Other activity
Apart from the summarized pharmacological activities mentioned above, the isolated constituents or crude extracts of Euodiae Fructus also involve other bioactivities including anti-diarrheal effect, antiallergic effect, antianoxic activity, antidepressant-like activity, antiviral activity, anti-ovotoxicity effect, etc. It has been reported that Euodiae Fructus has both anti-transit and anti-diarrheal effects with comparable ID 50 (the dose for 50% inhibition) values of 54 ± 7 and 76 ± 17 mg/kg and the anti-diarrheal effect of Euodiae Fructus may be associated with its anti-transit [102]. In vitro and vivo, Euodiae Fructus and its constituents (evodiamine and rutaecarpine) might inhibit the biosynthesis of anaphylaxis-related cytokines (TNF-α and IL-4) in mast cells and basophils, suggesting that they might be effective for IgE-induced allergic diseases such as atopic dermatitis and rhinitis [214]. Other studies have demonstrated that the involvement of cholinergic mechanism plays important roles in the antianoxic potential of evodiamine in the KCN-induced anoxia model [213,234]. Moreover, the antidepressant-like effect of evodiamine on chronic unpredictable mild stress rats probably by modulating effects on the monoamine transmitters and brain-derived neurotrophic factor (BDNF)-tropomyosin-related kinase B receptor (TrkB) signaling in the hippocampus [211]. Dai et al. showed that evodiamine could significantly inhibit the replication of anti-influenza A virus (IAV), the accumulation of LC3-II, p62 and EGFP-LC3, the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-α, IL-1β, IL-6 and IL-8 after IAV infection, meanwhile, the inhibition of IAV-induced autophagy by evodiamine was also dependent on its action on the AMPK/ TSC2/mTOR signal pathway [215]. In addition, the water extract of Euodiae Fructus could activate Akt to protect ovary cells against 4-vinylcyclohexene diepoxide-induced ovotoxicity, which indicates that Euodiae Fructus may help prevent premature ovarian failure or unexplained infertility caused by environmental factors [13]. Interestingly, a recent study has shown that aqueous extract of Euodiae Fructus and evodiamine could improve caffeineinduced sleep and excitation behaviors, at least in part, through the γ-aminobutyric acid (GABA) A -ergic system, these results suggest a potential therapeutic agent to treat insomnia or sleep problems related to caffeine intake [235].

Toxicity
According to China's most ancient herbal medicine book "Shen Nong's Herbal Classic" and 2020 Edition of Chinese Pharmacopoeia, the mild toxicity of Euodiae Fructus has been noted. In recent years, it has been reported that the cases of patients with chronic esophagitis, excessive use of Euodiae Fructus could cause stomach pain, vomiting, blurred vision and other toxic symptoms [236,237], and cause liver toxicity to the human body [238,239]. Modern researches in vitro and in vivo have shown that the crude extract and several compounds isolated from Euodiae Fructus have been reported to exert hepatic injury, CYP inhibition, and to induce proarrhythmic cardiotoxicity when used in high doses as described in Table 5, and the details will be further discussed below. In acute toxicity test, histopathological analysis revealed that Euodiae Fructus caused morphological changes in the liver, but no other main organs [240]. Cai et al. reported that oral gavaging of water decoction at dose of 6, 12 and 24 g/kg for 15 days in rats could increase malondialdehyde (MDA) level, and decrease the MnSOD activity and glutathione (GSH) levels reduction, followed by causing oxidative damage, finally resulting in adenosine triphosphate (ATP) depletion and cytochrome C (CytC) release, triggering cell death signaling pathways, which are all partial hepatotoxicity mechanisms of Euodiae Fructus [241], In another study, rutaecarpine might be a mechanism-based inhibitor of CYP1A2, and its potential hepatotoxicity might be related to reactive metabolites, and GSH trapping might be a detoxication route [242]. Furthermore, in vitro, rutaecarpine, evodiamine, and dehydroevodiamine significantly activated aryl hydrocarbon receptor (AHR), with an efficacy order of rutaecarpine > dehydroevodiamine > evodiamine, and ligand-docking analysis predicted that the methyl substitute at the N-14 atom was a key factor affecting AHR activation. The above three indole alkaloids were not hepatotoxic in vivo at the doses used. However, rutaecarpine and dehydroevodiamine disrupted bile acid homeostasis in an AHR-dependent manner, evodiamine failed to activate AHR due to its poor absorption in mice [243]. A recent study has revealed that rutaevin was shown to increase the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice serum, suggesting the potential hepatotoxicity of rutaevin, and the potential mechanism was that rutaevin was converted into a electrophilic BDA intermediate by CYP3A4 [244]. Moreover, it has been reported that dihydrorutaecarpine (5), 6-acetoxy-5-epilimonin (146) (72), and 1-methyl-2-tetradecy-4(1H)-quinolone (89)) [60], are speculated as possible hepatotoxic components based on spectrumtoxicity relationship and UPLC-Q-TOF-MS, whether these components were toxic as well still requires further exploring and researching. Therefore, attention should be given to monitoring bile acid metabolism in the clinical use of Euodiae Fructus.
It was worth noting that P450-mediated dehydrogenation reactions of evodiamine and rutaecarpine might cause toxicities through the generation of highly electrophilic intermediate and lead to drug-drug interactions mainly via the inactivation of CYP3A4 [246], Zhu et al. demonstrated that the induction of cytochrome P450 enzyme genes, hepatic transporters and phase-2 enzyme genes are involved in the interaction between rutaecarpine and drugs [247]. In addition, evodiamine could inhibit CYP1A2, CYP2C9 and CYP2D6 in rats, which might affect the disposition of drugs that rely on these pathways [248]. Therefore, it is necessary to pay attention to CYP3A4-, CYP1A2-, CYP2C9-and CYP2D6-mediated herb-drug interactions between Euodiae Fructus and western drugs to avoid undertreatment.
Additionally, dehydroevodiamine inhibited hERG channels with IC 50 values of 253.2 ± 26.3 nM on human embryonic kidney cells, prolonged the action potential duration (APD) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in a concentration-dependent manner from 0.01 to 1 μM and induced early afterdepolarizations (EADs) at 3 μM. Dehydroevodiamine (0.5 mg/kg) induced TdP arrhythmias in 2 out of 8 animals, and STV increased accordingly [249] in rabbits. In another study, evodiamine inhibited rat cardiomyocytes viability with IC 50 value of 28.44 µg/ mL at 24 h, increased LDH release and MDA levels, and reduced superoxide dismutase (SOD) activity on primary cultured neonatal rat cardiomyocytes. In zebrafish model, evodiamine also has a 10% lethal concentration of 354 ng/mL and induce cardiac malfunction, as evidenced by changes in heart rate and circulation, and pericardial malformations. These results indicated that evodiamine could cause cardiovascular side effects involving oxidative stress [250]. Since Euodiae Fructus contains potentially toxic compounds, reliable analytical methods are needed to control the quality of product development to ensure that the potential toxic components of Euodiae Fructus-related products are kept below allowable levels, and more attention should be given to herb-drug interactions and monitoring bile acid metabolism in the clinical use of Euodiae Fructus.

Quality control
As we all know, the intrinsic quality of TCM might vary greatly due to different geographic conditions and harvest periods [251]. Therefore, an efficient, rapid, sensitive and reproducible detection method was important to ensure the quality of each batch of medicinal materials [252]. According to the 2020 Edition of Chinese Pharmacopoeia, the concentration of evodiamine and rutaecarpine should exceed 0.15%, and the concentration of limonin should exceed 0.20% as determined by HPLC with the mobile phase making up of 0.02% phosphoric acid water and acetonitrile-tetrahydrofuran (25:15) at a ratio of 35: 65, and the detection wavelength should be at 225 nm. However, due to the pharmacological activity and toxicity of multiple ingredients mentioned above, the content of single or small amount of labeled compounds cannot accurately reflect the quality of TCM [253]. With the advancement of analytical tools, it is necessary to adopt more advanced detection methods to qualitatively and quantitatively analyze as many biologically active ingredients as possible. A total of 13 compounds: Wuchuyuamide-I, quercetin, limonin, evodiamine, rutaecarpine [254], dehydroevodiamine, evodine [26], evodiamide, 14-formyldihydrorutaecarpine [25], 1-methyl-2-undecyl-4(1H)quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine [255], were selected to ensure the quality of Euodiae Fructus by HPLC-DAD, HPLC-DAD-MS/MS, HPLC/UV/APCI-MS/MS, and CEC-MS, and the additional details are listed in Table 6.
To evaluate the quality, the newly established fingerprint analysis was conducted on this kind of plants. The fingerprint analysis of Euodiae Fructus was carried out and the results suggest that the chemical components would vary greatly in different locations and vary a little in different years in the same site [64,256]. In recent years, one study compared the differences of essential oils from three species of Euodiae Fructus cultured in China. The results showed that the differences in chemical composition and oil production within species are greater than the differences between species [91].

Conclusions
This review has summarized the multifaceted uses and recent findings regarding studies of the phytochemistry, traditional use, bioactive constituents, pharmacology, toxicity, and quality control of different extracts and compounds of Euodiae Fructus and provides a practical base for further scientific research and favorable clinical application on this plant. Extensive researches have been conducted on the phytochemistry of the Euodiae Fructus and approximately 240 compounds have been isolated and identified from this plant, including alkaloids, terpenoids, steroids, phenols, volatile oil and other compounds. As the literature has demonstrated, alkaloids and terpenoids are the main components of Euodiae Fructus, and alkaloids are mostly responsible for its pharmacological activities. Additionally, recent reports have primarily focused on evaluating anticancer, antibacterial, anti-inflammatory, insecticidal, anti-cardiovascular disease, neuroprotective, anti-obesity and anti-diabetic activities of the herbal medicines derived from this plant. In particular, the indole alkaloids (e.g., evodiamine, rutaecarpine and dehydroevodiamine) and limonin have been confirmed to has low toxicity and high medicinal value through various pharmacological activities in vivo and in vitro investigations.
Euodiae Fructus exhibits a diverse set of pharmacological properties and its chemistry is complex. For these reasons, it is of great importance to systematically and critically evaluate the future direction and application of this field. Although many efforts have been made to study these plants, there are also a number of points and aspects that need to be improved and researched further: (1) According to TCM, Euodiae Fructus is traditionally considered to have mild toxicity, and a few support studies have been linked to its toxicity, including the potential hepatotoxicity, CYP inhibition, and cardiotoxicity of this plant. Thus, it is necessary to investigate the potential toxic effects induced by Euodiae Fructus and clarify the toxic components, target-organs and mechanisms, so as to lay a foundation for future research. (2) Several traditional uses of these plants have been validated in recent pharmacological studies, but some of these were only tested in vitro. Therefore, the effectiveness of these compounds in vivo and comprehensive placebo-controlled and double-blind clinical trials need to be further studied, and more detailed pharmacology and mechanism of action may help to better understand TCM theory. (3) Alkaloids are traditionally considered as the major bioactive compounds in Euodiae Fructus. However, their mechanisms of action remain unclear, and further studies are required to understand the structure-activity relationships of these constituents and bioactivities. For isolated alkaloids, too many researches are focused on evodiamine and rutaecarpine, and there are other active ingredients  [254] Dehydroevodiamine, Evodine, Evodiamine and Rutaecarpine HPLC-DAD-MS/MS 0.10-0.51%, 0.49-3.12%, 0.07-1.56%, and 0.10-0.69%, respectively (contents) [26] Rutaecarpine, Evodiamine, Evodiamide, 14-Formyldihydrorutaecarpine, Dehydroevodiamine HPLC/UV/APCI-MS/MS 0.061-0.550 mg/g, 0.039-1.623 mg/g, 0.0037-0.455 mg/g, 0.024-0.065 mg/g, 0.122-0.863 mg/g and 0.0069-0.741 mg/g, respectively(contents) [25] Limonin, Evodiamine, Rutaecarpine, 1-Methyl-2-undecyl-4(1H) quinolone, Evocarpine, 1-Methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone and Dihydroevocarpine like dehydroevodiamine, evocarpine and dihydroevocarpine, etc. that have been lacked of research or ignored. Further investigation should be encouraged to study these components or their analogues. (4) Numerous studies have demonstrated evodiamine process extensive activities, however, due to its poor water solubility and low oral bioavailability, thereby limiting its anticancer efficacy clinically. Future studies should aim to overcome these problems in the clinical application of TCM. (5) In view of the toxicity of some compounds, reliable analytical methods are required for proper quality control of drug development to ensure that potential toxic components remain below the tolerance level of Euodiae Fructus.