The Ethnopharmacological, Phytochemical, and Pharmacological Review of Euryale ferox Salisb.: A Chinese Medicine Food Homology

Euryale ferox Salisb. (prickly water lily) is the only extent of the genus Euryale that has been widely distributed in China, India, Korea, and Japan. The seeds of E. ferox (EFS) have been categorized as superior food for 2000 years in China, based on their abundant nutrients including polysaccharides, polyphenols, sesquineolignans, tocopherols, cyclic dipeptides, glucosylsterols, cerebrosides, and triterpenoids. These constituents exert multiple pharmacological effects, such as antioxidant, hypoglycemic, cardioprotective, antibacterial, anticancer, antidepression, and hepatoprotective properties. There are very few summarized reports on E. ferox, albeit with its high nutritional value and beneficial activities. Therefore, we collected the reported literature (since 1980), medical classics, database, and pharmacopeia of E. ferox, and summarized the botanical classification, traditional uses, phytochemicals, and pharmacological effects of E. ferox, which will provide new insights for further research and development of EFS-derived functional products.

As a folk medicine in China for thousands of years, EFS is primarily used to reinforce the kidney, invigorate essence, and tonify the spleen to arrest diarrhea. It is commonly employed to manage conditions such as spermatorrhea, gonorrhea, dysmenorrhea, incontinence of urine, and diarrhea of the bowels [9]. In March 2002, the National Health Care Commission of the People's Republic of China embodied EFS as one of the new herbal medicines on the list of medicinal and food ingredients, with the stipulation that it can be used for both medicinal and food purposes within a limited range and dosage. The dried ripe seed is included in the Chinese Pharmacopoeia (2020 Edition) as a commonly used Chinese herbal medicine. According to the theory of Chinese medicine, it tastes sweet,   Eliminating dampness, easing backache and knee pain, tonifying and removing malignant diseases, benefiting the essence, strengthening the will, and making the ears and eyes wise. [5] The Compendium of Materia Medica (Bĕn Căo Gāng Mù, 本草纲目) Ming Dynasty, AD 1578, China Quenching thirst and benefiting the kidney, treating urinary incontinence, spermatorrhea, and leucorrhea. [28] The Song of Medicinal Properties and four hundred flavours (Yào Xìng Gē Kuò Sì Băi Wèi Bái Huà Jiě, 药性歌括四百味) Ming Dynasty, AD 1581, China Benefits the essence, relieving soreness of the waist and knees, and arresting seminal emission. [29] Leigong Concocted Medicinal Annotation (Léi Gōng Páo Zhì Yào Xìng Jiě, 雷公炮制药性解) Ming Dynasty, AD 1622, China Tonifying the spleen and stomach, benefitting the essence, improving visual and auditory acuity, and amnesia. [29] Essentials of Chinese Materia Medica (Běn Căo Bèi Yào, 本草备要) Kangxi XXXIII, AD 1694, China Strengthening the kidney and benefiting the essence, tonifying the spleen, and eliminating dampness. Treating diarrhea with turbidity and spermatorrhea. [29] Chinese Pharmacopoeia AD 2020, China Benefiting the kidney and consolidating sperm, tonifying the spleen and inhibiting diarrhoea, eliminating dampness and arresting leucorrhea, improving spermatorrhea, enuresis and frequent urination, splenoasthenic diarrhea, and leucorrhea. [9] Traditional Medical & Pharmaceutical Database Japan Improving metabolic arthritis, urinary incontinence, and leucorrhea, easing waist pain. -Ayurveda and Unani system India Improving rheumatic and bile disorders, against dysmenorrhea and exerting spermatogenic properties. [27] Stems The Compendium of Materia Medica (Bĕn Căo Gāng Mù, 本草纲目) Ming Dynasty, AD 1578, China Quenching irritability and thirst, eliminating asthenia-heat syndrome. [28] Leaves The Compendium of Materia Medica (Bĕn Căo Gāng Mù, 本草纲目) Ming Dynasty, AD 1578, China Treating retained placenta and haematemesis. [28] Roots The Compendium of Materia Medica (Bĕn Căo Gāng Mù, 本草纲目) Ming Dynasty, AD 1578, China Improving swollen testicles, and abdominal pain due to stagnation of vital energy. [28]

Phytochemistry
Various phytochemicals have been isolated and determined in E. ferox, which can be classified into polysaccharides, polyphenols, flavonoids, cyclic dipeptides, cerebrosides, phytosterol, tocopherols, and triterpenoids based on the chemical properties. Over 100 secondary constituents were tentatively identified from leaves, petioles, fruit peels, seed shells, and kernels of E. ferox by UHPLC-MS/MS analysis, with polyphenols occupying 87%. Glycosylated flavonoids are significantly accumulated in the leaves, polyphenols are abundant in the seed, shell, and phenolic acids are predominant in the fruit peel. However, flavonoids vary among the five tissues [30]. All of those reported phytoconstituents are listed in Table 2 in text and their structures are presented in Figures 2-9.

Polysaccharides
Polysaccharides are one of the main components of E. ferox that exert multiple pharmacological activities [10]. A polysaccharide named EPJ (1) was isolated from EFS by DEAE-52 cellulose chromatography and Sephadex G-100 column, which is mainly composed of glucose and rhamnose with a molar ratio of 5.46:1, and the molecular weight was determined to 15.367 kDa [31]. Zhang et al. isolated a novel polysaccharide EFSP-1 (2) from EFS by DEAE sepharose FF and Superdex™ 75 gel chromatography, which was mainly composed of (1→4)-α-D-Glcp with branches substituted at O-6 and terminated with T-α-D-Glcp. The structure of EFSP-1 was characterized by NMR, FT-IR, and GC-MS [32]. The high starch content (72.27-83%) made E. ferox into a superfood, while resistant starch has gained widespread focus for its physiological functions. A type 3 resistant starch (RS3) was isolated from EFS, and it belongs to B + V type crystal and exerts high thermal stability [33]. Moreover, amylopullulanase-treated E. ferox flour promoted the content of resistant starch and inhibited the release of glucose during in vitro digestibility analysis [34].

Polyphenols and Flavonoids
Polyphenols are a class of secondary metabolites with a polyphenolic structure widely present in E. ferox, mainly existing in the seed coat, roots, leaves, and fruits. An ultrasonicassisted extraction technology was performed for the extraction of phenolic compounds from E. ferox seed shells, and three polyphenols and one flavonoid were determined by HPLC analysis (3-5, 12) ( Table 2). In addition, resveratrol (6), compound 4-O-methyl gallic acid (7), and protocatechuic acid (8) were identified from the ethyl acetate extract of EFS. Dihydroflavonoids are the most reported flavonoids in the seeds of E. ferox (9)(10)(11). The targeted flavonoid metabolome was determined to explore the dynamic changes of flavonoid biosynthesis by LC-ESI-MS/MS analysis, a total of 129 flavonoid metabolites were identified, including 11 flavanones, 8 dihydroflavanols, 16 flavanols, 29 flavonoids, 3 isoflavones, 12 anthocyanins, 29 flavonols, 6 flavonoid carbosides, 3 chalcones, and 13 proanthocyanidins [35]. However, these compounds were inferred by mass spectrometry and their reliability needs to be further verified. The structures of isolated polyphenols and flavonoids are shown in Figure 2.

Cyclic Peptides
Cyclic peptides are a kind of cyclo-compounds composed of common and uncommon amino acids. Due to specific properties, such as good target selectivity, binding affinity, and low toxicity, cyclic peptides have become attractive lead compounds for drug development [50]. Multiple bioactive activities have been reported including antimicrobial, anti-infection, anti-tumors, anti-chronic kidney diseases, anti-diabetes, and memory improvement [51,52]. Using thin-layer in situ chemical reactions, several cyclic dipeptides (13)(14)(15)(16)(17)(18) have been isolated from EFS. The isolated cyclic peptides are shown in Table 2 and Figure 3.

Cyclic Peptides
Cyclic peptides are a kind of cyclo-compounds composed of common and mon amino acids. Due to specific properties, such as good target selectivity, bin finity, and low toxicity, cyclic peptides have become attractive lead compounds f development [50]. Multiple bioactive activities have been reported including an bial, anti-infection, anti-tumors, anti-chronic kidney diseases, anti-diabetes, and m improvement [51,52]. Using thin-layer in situ chemical reactions, several cyclic dip (13)(14)(15)(16)(17)(18) have been isolated from EFS. The isolated cyclic peptides are shown in Tab Figure 3.

Cerebrosides
Cerebrosides are neutral chemicals that consist of a monosaccharide and ce bound by a β-glycosidic bond to the C1 of esfingol. As an important componen membranes in the nervous system, cerebrosides play critical roles in regulating me dynamics and forming internal structures. Four novel cerebrosides have been elu in the rhizome with the adventitious root of E. ferox (19,20) and EFS (21, 22), resp [41,42]. The structures of cerebrosides are shown in Figure 4.

Cerebrosides
Cerebrosides are neutral chemicals that consist of a monosaccharide and ceramide bound by a β-glycosidic bond to the C1 of esfingol. As an important component of cell membranes in the nervous system, cerebrosides play critical roles in regulating membrane dynamics and forming internal structures. Four novel cerebrosides have been elucidated in the rhizome with the adventitious root of E. ferox (19,20) and EFS (21,22), respectively [41,42]. The structures of cerebrosides are shown in Figure 4.
Triterpenoids are of great interest to researchers owing to their wide range of biolog ical activities. Gong determined the contents of triterpenoids in 70% ethanol extract of E ferox seed shell using vanillin-perchloric acid method, the total triterpenoids are up to 36.7% [53]. In order to investigate the putative active compounds responsible for antidia betic, antioxidant, and antihyperlipidemic in EFS, Ahmed et al. obtained two nove triterpenoids, 2β-hydroxybetulinic acid 3β-oleiate (29) and 2β-hydroxybetulinic acid 3β caprylate (30), from the ethyl acetate extract [44,45]. The specific information and struc tures of each compound are shown in Table 2, Figure 5.
Triterpenoids are of great interest to researchers owing to their wide range of biological activities. Gong determined the contents of triterpenoids in 70% ethanol extract of E. ferox seed shell using vanillin-perchloric acid method, the total triterpenoids are up to 36.7% [53]. In order to investigate the putative active compounds responsible for antidiabetic, antioxidant, and antihyperlipidemic in EFS, Ahmed et al. obtained two novel triterpenoids, 2β-hydroxybetulinic acid 3β-oleiate (29) and 2β-hydroxybetulinic acid 3βcaprylate (30), from the ethyl acetate extract [44,45]. The specific information and structures of each compound are shown in Table 2, Figure 5.
Triterpenoids are of great interest to researchers owing to their wide range of biological activities. Gong determined the contents of triterpenoids in 70% ethanol extract of E. ferox seed shell using vanillin-perchloric acid method, the total triterpenoids are up to 36.7% [53]. In order to investigate the putative active compounds responsible for antidiabetic, antioxidant, and antihyperlipidemic in EFS, Ahmed et al. obtained two novel triterpenoids, 2β-hydroxybetulinic acid 3β-oleiate (29) and 2β-hydroxybetulinic acid 3βcaprylate (30), from the ethyl acetate extract [44,45]. The specific information and structures of each compound are shown in Table 2, Figure 5.

Tocopherol
Tocopherols are a series of organic chemicals consisting of various methylated phenols. EFS contains an extraordinarily high content of tocopherols, which may contribute to scavenging free radicals and antioxidant effects. After extraction with 95% methanol
OR PEER REVIEW 15 of 2 methoxybenzyl]tetrahydrofuran (48), were also isolated from EFS [37,38]. The isolated lignans are shown in Table 2, and the corresponding structures are shown in Figure 7.

Volatile Constituents
A Clevenger's apparatus was used for the isolation of essential oil by hydro-distilla tion for 6 h in EFS with a yield of 0.028% (v/w). A total of 37 components were identified by gas chromatography-mass spectroscopy. Compounds were identified by comparison of their retention indices and mass spectra with the data stored in the National Institute of Standards and Technology (NIST 05). The main constituents were butylated hydroxy toluene (49) (38.7%), palmitic acid (50) (11.0%), linoleic acid (51) (9.0%), and hexanoic acid (53) (3.9%) [46]. The structures of volatile constituents (49-83) are shown in Figure 8.

Volatile Constituents
A Clevenger's apparatus was used for the isolation of essential oil by hydro-distillation for 6 h in EFS with a yield of 0.028% (v/w). A total of 37 components were identified by gas chromatography-mass spectroscopy. Compounds were identified by comparison of their retention indices and mass spectra with the data stored in the National Institute of Standards and Technology (NIST 05). The main constituents were butylated hydroxytoluene (49) (38.7%), palmitic acid (50) (11.0%), linoleic acid (51) (9.0%), and hexanoic acid (53) (3.9%) [46]. The structures of volatile constituents (49-83) are shown in Figure 8.  (Figure 9). An ultrasound-assisted technique was established for the extraction of anthocyanins from the waste leaves of E. ferox, and the yield of anthocyanins was 2.82 ± 0.03 mg/g. Nineteen anthocyanins were identified by HPLC-QTOF-MS/MS [26].

Pharmacological Activities
Various in vitro and in vivo studies have indicated that E. ferox-derived extracts and phytoconstituents exhibited antioxidant and antidiabetic effects. In addition, the anti-tu mor, anti-hyperlipidaemic, antibacterial, anti-inflammatory, antimelanogenic, antiaging antifatigue, cardioprotective, and hepatoprotective activities have also been reported. Th pharmacological properties of E. ferox were summarized in Table 3 and Figure 10.

Pharmacological Activities
Various in vitro and in vivo studies have indicated that E. ferox-derived extracts and phytoconstituents exhibited antioxidant and antidiabetic effects. In addition, the anti-tumor, anti-hyperlipidaemic, antibacterial, anti-inflammatory, antimelanogenic, antiaging, antifatigue, cardioprotective, and hepatoprotective activities have also been reported. The pharmacological properties of E. ferox were summarized in Table 3 and Figure 10.

Antioxidant and Anti-Inflammatory Activity
The evaluation of antioxidant activity can be performed in three main ways including directly 1,1-diphenyl-2-picrylhydrazyl (DPPH) and reactive oxygen species (ROS) scavenge, the activation of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and improvement of somatic cellular integrity. The methanol, ethanol, and aqueous extracts of EFS showed DPPH scavenging effects, while the methanol extract showed anti-inflammatory activity in RAW 264.7 cell lines [19][20][21]39]. A corilagin monomer was isolated and identified from E. ferox shell, and showed an anti-inflammatory effect against LPS-induced Raw264.7 cells, the level of NO, TNF-α, IL-6, and IL-1β were significantly reduced, while the mechanism was related to NF-κB and MAPK signaling pathway [17].
EFS methanol extracts exerted high levels of DPPH radical scavenging activity, lipid peroxidation inhibition, protection of H2O2-induced apoptosis, and antioxidant enzyme activity enhancement. Among various fractionated samples of E. ferox, the ethyl acetate and butanol fractions exhibited relatively high antioxidative activity [20]. The essential oil from the EFS exhibited strong DPPH and ABTS scavenging activity, the IC50 of which were 6.27 ± 0.31 and 2.19 ± 0.61 μg/mL, respectively [46]. Fermentation of E. ferox with Lactobacillus curvatus increases the content of the various bioactive components including smaller molecular weights of polysaccharides and polypeptides, enhances its antioxidant capacity, and attenuates oxidative stress-induced human skin fibroblast apoptosis and senescence [21]. In addition, the phenolic extracts from the E. ferox seed shells and anthocyanins from the E. ferox leaves showed DPPH and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging effects [18,26,36].
The anti-oxidative activities vary among different parts of E. ferox, while the seed extracts showed better effects than seed shells, leaves, petioles, and pedicels from the

Antioxidant and Anti-Inflammatory Activity
The evaluation of antioxidant activity can be performed in three main ways including directly 1,1-diphenyl-2-picrylhydrazyl (DPPH) and reactive oxygen species (ROS) scavenge, the activation of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and improvement of somatic cellular integrity. The methanol, ethanol, and aqueous extracts of EFS showed DPPH scavenging effects, while the methanol extract showed anti-inflammatory activity in RAW 264.7 cell lines [19][20][21]39]. A corilagin monomer was isolated and identified from E. ferox shell, and showed an anti-inflammatory effect against LPS-induced Raw264.7 cells, the level of NO, TNF-α, IL-6, and IL-1β were significantly reduced, while the mechanism was related to NF-κB and MAPK signaling pathway [17].
EFS methanol extracts exerted high levels of DPPH radical scavenging activity, lipid peroxidation inhibition, protection of H 2 O 2 -induced apoptosis, and antioxidant enzyme activity enhancement. Among various fractionated samples of E. ferox, the ethyl acetate and butanol fractions exhibited relatively high antioxidative activity [20]. The essential oil from the EFS exhibited strong DPPH and ABTS scavenging activity, the IC 50 of which were 6.27 ± 0.31 and 2.19 ± 0.61 µg/mL, respectively [46]. Fermentation of E. ferox with Lactobacillus curvatus increases the content of the various bioactive components including smaller molecular weights of polysaccharides and polypeptides, enhances its antioxidant capacity, and attenuates oxidative stress-induced human skin fibroblast apoptosis and senescence [21]. In addition, the phenolic extracts from the E. ferox seed shells and anthocyanins from the E. ferox leaves showed DPPH and 2,2 ′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging effects [18,26,36].
Cell wall polysaccharides (EFPP) were isolated from the petioles and pedicels of E. ferox using the DEAE-52 column, and four major fractions (EFPP-1, EFPP-2, EFPP-3, and EFPP-4) were obtained. The crude EFPP and EFPP-4 could effective against H 2 O 2 -induced injury on HUVEC and VSMC through enhancement of T-AOC, SOD, and CAT activities and decrease of MDA content [55].
The anti-oxidative activities vary among different parts of E. ferox, while the seed extracts showed better effects than seed shells, leaves, petioles, and pedicels from the aforementioned reports. Further studies should aim to purify and characterize the active phytoconstituents from the antioxidative extracts.

Antidiabetic and Hypoglycemic Activity
The E. ferox ethanol extract protected β-cells against ROS-mediated damage by increasing the expression of antioxidant enzymes and reducing hyperglycemia, possibly due to the release of insulin from residual and recovered β-cells in the pancreas of streptozotocin-induced diabetic rats [15]. Another study indicated that germinated EFS extract contained more gentisic acid, caffeic acid, and other 27 effective polyphenols than EFS, corresponding to the higher improved antioxidant and renal indexes, and a more stable effect in regulating the AMPK/mTOR and Keap1/Nrf2/HO-1 signaling pathways, leading to the more attenuated antidiabetic effects [56]. A polysaccharide obtained from EFS, EFSP-1, could increase glucose consumption by up-regulating the expression of GLUT-4 via activating PI3K/Akt signal pathway in insulin resistance HepG2 and 3T3-L1 cells [32]. The antidiabetic activities of two triterpenoids in E. ferox were investigated in streptozotocininduced Wistar rats over a four-week period. After 45 days of gavage consisting of 2βhydroxybetulinic acid 3β-caprylate (HBAC) and 2β-hydroxybetulinic acid 3β-oleiate (HBAO) in diabetic mice, the plasma glucose and insulin were normalized, pancreatic βcell, the histological architecture of pancreas, kidney, and liver were restored, as well as the endogenous antioxidant enzymes [44,45]. The aforementioned studies suggest that extract of EFS could be an important source of natural antioxidants with hypoglycaemic and hypolipidaemic effects, and could be used as a food additive or functional food in the future.
Another study investigated the extract of Gorgon fruit as a food additive and found that E. ferox shell extract (EFSSE) had a significant effect on the in vitro digestibility of bread starch and that EFSSE (2%) fortified bread and exhibited a strong glycemic index inhibition. In addition, the IC 50 of EFSSE on α-amylase and α-glucosidase inhibitory effect was 62.95 and 52.06 µg/mL, respectively [57]. The hypoglycemic and hypolipidemic effects of triterpenoid-rich 75% ethanol extracts of E. ferox shell were investigated in streptozotocininduced diabetic mice. Gavage of 400 and 600 mg/kg E. ferox shell extract for 4 weeks significantly restored the body weight, blood glucose, and insulin resistance [53]. Triterpenoidrich E. ferox shell extract in drinking water (500 mg/L) for 4 weeks significantly attenuated streptozotocin-induced high blood glucose, pancreas injury, higher tyrosine phosphatase-1B level, and low insulin receptor substrate expression [58]. Therefore, E. ferox shell extract can be used as a therapeutic ingredient for diabetes induced by insulin resistance.
Crude polysaccharides (EFPP) were prepared from the petioles and pedicels of E. ferox, which had a total carbohydrate of 65.72 ± 2.81%, the monosaccharide compositions were Man, GlcA, Rha, Glc, Gal, and Ara at a molar ratio of 0.12:0.01:9.57:0.41:1.00:0.24. After oral administration with EFPP (400 mg/kg) for 28 days, the activities of CAT, SOD and GSH-Px, and MDA contents in the kidney and liver of alloxan-induced mice were significantly ameliorated, as well as the damaged pancreas, kidney, and liver tissues. The blood glucose level was reduced and the serum insulin level was remarkably increased [60].
Currently, network pharmacology as an emerging discipline has been gradually applied to the mechanistic study of phytopharmaceuticals. This method is suitable for multicomponent and multi-target studies by using the database of ingredients, targets, and genes to elucidate the complex mechanism of action of a drug in a holistic way. Some investigations have preliminarily elucidated the anti-diabetic mechanism of action of E. ferox based on network pharmacology and molecular docking. Twenty-four components of E. ferox and 72 targets were identified, of which 9 (FABP1, JUN, LPL, PPARA, TP53, TGFB1, IL1A, MAPK1, CTNNB1) are clinically relevant and mainly regulated by transcription factors such as HNF4A and PPARG. The main components are oleic acid, which targets the proteins encoded by PPARA, LPL, and FABP1, and vitamin E, which binds to the proteins encoded by MAPK1 and TGFB1 [59].
In conclusion, E. ferox can be used to treat diabetes mainly through anti-inflammatory, reducing pancreatic β-cell damage and apoptosis, promoting glucose absorption and utilization, and improving insulin resistance and complications. Although noteworthy antidiabetic properties have been attributed to E. ferox polysaccharides or triterpenoids, the homopolysaccharide has not been identified, and whether there are any other phytoconstituents responsible for this activity remains to be elucidated. Meanwhile, further clinical validation of the above findings is still needed in conjunction with experiments.

Hepatoprotective and Cardioprotective Activity
Oral administration of the E. ferox seed coat ethanol extract (EFSCE) to high-fat diet (HFD)-induced ICR mice at doses of 15 and 30 mg/kg for 4 weeks resulted in a significant reduction in body weight, lipid deposition in the liver and blood lipids. EFSCE also prevented excessive production of MDA and enhanced SOD activity to counteract oxidative stress. In addition, EFSCE was effective in reducing alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in HFD-induced mice. EFSCE can be used as a biologically active natural product for the treatment of HFD-induced NAFLD by modulating IRs-1 and CYP2E1 to eliminate lipid accumulation and oxidative stress [13].
Another study investigated if E. ferox seeds could reduce myocardial ischemic reperfusion injury. The isolated rat hearts ischemia and reperfusion acute model was constructed to evaluate the cardioprotective effect of E. ferox extract (25,125 or 250 µg/mL), 125 or 250 µg/mL E. ferox extract treatment significantly enhanced aortic flow and reduced the infarct size. E. ferox (250 and 500 mg/kg/day) oral administration for 21 days improved postischemic ventricular function and reduced myocardial infarct size in a chronic ischemic reperfusion model. Two cardioprotective proteins, TRP32, and thioredoxin, were significantly increased. Taken together, this study demonstrated the cardioprotective properties of Makhana and the effects may be related to its upregulation of TRP32 and Trx-1 proteins and ROS scavenge activities [14].

Cytotoxic and Anticancer Activity
The apoptotic effects of EFS ethanol extract (ESE) in A549 lung cancer cells were investigated, ESE induces apoptosis via the regulation of mitochondrial outer membrane potential and generation of ROS. ESE-induced A549 apoptosis is in a p53-dependent manner, in addition, ESE suppressed tumor growth in Balb/c-nu mice bearing A549 xenografts and activated p53 protein [16]. E. ferox seed shell extracts (200 µg/mL) showed an inhibitory effect on SGC7901 and HepG2 cell proliferation, with the inhibition rate being 92.63% and 72.40%, respectively. E. ferox seed shell extracts (200-800 µg/mL) arrest SGC7901 cells in the G0/G1 phase, and 50-200 µg/mL E. ferox seed shell extracts arrest HepG2 cells in the S phase. Meanwhile, the cell mitochondrial membrane potential was significantly reduced and the intracellular calcium influx was increased [61]. Treatment of melan-a cells with 30 µg/mL EFS ethyl acetate fraction produced a strong inhibition of cellular tyrosinase and melanin synthesis, and the lysosomal degradation of tyrosinase was involved in melanogenesis inhibition [23]. Resorcinol (95) inhibited melanin synthesis in B16F10 melanoma cells with an IC50 value of 492.8 µM [37].
Two cerebrosides, ferocerebrosides A and B, were isolated from the methanol extract of EFS, and they showed marginal toxicity against brine shrimp with LC50 values of 0.17 and 0.20 mM, respectively [42]. The toxicity study of a new glucan EFSP-1, obtained from EFS, was performed on HepG2 and 3T3-L1 cells, and no obvious toxicity was observed at doses between 100 and 400 µg/mL [32]. Neuroprotective effect of EFS subfractions against glutamate-induced cytotoxicity in hybridoma cells N18-RE-105 was investigated. The EFS ethanolic extract showed a dose-dependent protective effect against 20 mM glutamateinduced neuronal cell death. EFS ethanolic extract was subfractionated with hexane, diethyl ether, and ethyl acetate, the hexane fraction showed the strongest neuroprotective effect against glutamate-induced N18-RE-105 cells. The results suggest that EFS can be used as chemotherapeutic agents in the treatment of neurological disorders [62].

Antifatigue Activity
An exertional swimming test was performed to evaluate the anti-fatigue effect. The phenolic extracts of E. ferox can prolong the average duration of exertional swimming, the expression of BUN was significantly reduced, while hepatic glycogen content was dramatically increased. In addition, three main phenolic compounds in the extract were identified as 5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-chroman-4-one, naringenin, and buddlenol E [18].
Studies have shown that E. ferox is a potential and readily available source of natural antioxidants and has the potential to be a new functional anti-fatigue food or drug. In the future, studies on the chemical composition and safety evaluation of phenolic extract need to be continued with a view to providing valuable information for novel functional food development.

Anti-Depressant Activity
The potential antidepressant effects of EFS petroleum ether fraction (ES-PE) were investigated in a mouse model of chronic unpredictable mild stress (CUMS). Deficits in the open field test, sucrose preference test, tail suspension test, and forced swimming test were observed in mice following CUMS and were reversed following ES-PE administration. ES-PE significantly up-regulated phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and mammalian autophagy initiating kinase (ULK1) at Ser317, and the ration of p-mTOR/mTOR was suppressed by ES-PE treatment. In addition, ES-PE treatment significantly attenuated Compound C, an inhibitor of AMPK, induced autophagy suppression. GC-MS analysis revealed high levels of vitamin E acetate in ES-PE, suggesting the potential role of VE in the antidepressant effect of ES-PE [22]. Further studies are needed to explore the antidepressant mechanism of ES-PE, in addition to autophagy, as well as other potential phytochemicals.

Other Activities
E. ferox seed methanolic extracts exhibit significant antibacterial activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853, the minimum inhibitory concentration (MIC) was 64, 128, and 64 mg/L, respectively [63]. Using the agar cup method, ethyl acetate and ethanol extract of E. ferox seed coat showed a higher inhibition zone against E. coli and S. aureus [64]. In addition, the methanolic E. ferox seed and leaf extracts showed anti-fungal effects against Candida albicans and Pencillium notatum strains [65].

Toxicity
The non-toxic characteristic of EFS was clearly stated thousands of years ago in Shennong's Herbal Classic [5]. According to Chinese Pharmacopoeia, the medicinal dosage of EFS is generally 9-15 g per day [13]. If consumed excessively, it may lead to gastrointestinal overload, as EFS contains a lot of starch, protein, and other ingredients that have a solid and astringent effect [34,57]. The "Suixiju Dietary Recipes" records the contraindications of EFS as "EFS is not recommended in the following conditions, including before and after cold affection, malaria, dysentery and hemorrhoids, red urine and constipation, transport failure of the spleen, and postpartum period". In the modern toxicity evaluations, 24 h incubation of EFS essential oil showed toxicity with an LC50 value of 11.48 ± 0.51 µg/mL in brine shrimp bioassay [46]. Compounds 21 and 22 also showed cytotoxicity in the brine shrimp lethality bioassay, with LC50 values of 0.17 and 0.20 mM, respectively [42]. In another study, oral administration of 70% ethanol EFS extract to Wistar rats for 45 days at doses of 100-400 mg/kg, no lethality and toxicity were found [15]. Nevertheless, the monitoring of adverse reactions to EFS should be further strengthened to improve its safety in clinical application.

Conclusions and Perspectives
The current study summarized the investigations of E. ferox in terms of traditional uses, phytochemistry, pharmacological effects, and toxicity in recent decades. It is ex-pected to provide a preliminary basis for future research on E. ferox and to provide a reference for further studies on the biological activities and clinical applications.
Firstly, E. ferox contains complex and diverse chemical constituents, including triterpenes, sterols, flavonoids, phenylpropanoids, essential oils, organic acids, and polysaccharides. So far, more than 100 compounds have been isolated and identified. However, although many chemical components have been elucidated, only a few of them have been validated for their biological activity. There is a lack of in-depth studies on the mechanisms of physiological activity of polysaccharides. The homosaccharide, structural information, monosaccharide composition, and content need to be further investigated.
Secondly, the anti-diabetes, gastrointestinal diseases, and even anti-cancer effects of E. ferox have been greatly explored. However, the understanding of its mechanisms and pathways of action remains ambiguous and is mostly based on its anti-oxidant effects. In addition, there are few studies that addressed the toxicities of E. ferox, and the pharmacokinetics and drug interactions of E. ferox in vivo remain unknown.
Further research and development are necessary for the following aspects. Firstly, it is important to continue isolating, purifying, and identifying chemical components, with emphasis on the biological activity and structure-activity relationships. Secondly, more indepth studies should be conducted to determine the mechanisms of the physiological activity of polysaccharides present in E. ferox. This will shed more light on its biological activities and clinical applications. Thirdly, new techniques and methods such as molecular biology, cell biology, and histology should be combined to obtain intuitive evaluations via animal and clinical experiments and further explore intrinsic pharmacological mechanisms.
As a medicinal food ingredient with rich nutritional value and various health functions, E. ferox have good prospects for market development. The design of E. ferox special functional food and the utilization of its derived waste material might be the future directions.  Data Availability Statement: Data sharing not applicable to this article as no datasets were generated or analysed during the current study.