Pharmacological Activities of E. ferox: Comparison
Please note this is a comparison between Version 1 by Zhiyou Yang and Version 2 by Jason Zhu.

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. 

  • Euryale ferox
  • traditional medicine
  • phytochemical constituents

1. Introduction

Euryale ferox seed (EFS) is a typical representative of “a medicine food homology species”, as described in the Huangdi Nei Jing Tai Su (黄帝内经太素): “Eating it as food on an empty stomach, and taking it as medicine for patients” [1][2][1,2]. EFS is the dried seeds of the E. ferox Salisb. plant (Figure 1A), and is widely distributed in India, Bangladesh, Myanmar, New Zealand, Russia, Thailand, and parts of East Asia [3][4][3,4]. In China, EFS was first described in “Shen Nong’s Classic of the Materia Medica” (Shén Nóng Bĕn Căo Jīng, 神农本草经) [5]. EFS, also known as Foxnut, Lotus seeds, Gorgon nuts, and Phool Makhana, is spherical, commonly broken grains (Figure 1B) [6][7][8][6,7,8].
Figure 1. The biotope and morphological characteristic of E. ferox. (A) The leaves of E. ferox. (B) The seeds of E. ferox. (C) the pedicels, receptacles, sepals, and corolla of E. ferox. The fruits of Southern E. ferox (D) and northern E. ferox (E) are presented.
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, bitter, and astringent, and attributes to the spleen and kidney meridians [9]. The raw EFS comprises about 61% carbohydrates, 12.1% moisture, 15.6% protein, 1.35% fat, 7.6% fiber, and 1.8% minerals, and possesses a calorific value of 362 kcal/100 g [10]. EFS contains an assortment of chemical constituents including triterpenoids, sterols, flavonoids, phenylpropanoids, organic acids, essential oils, and polysaccharides, while triterpenoids and flavonoids are considered major active components [8][10][11][12][8,10,11,12]. Modern pharmacological studies indicate that it exerts a wide range of bioactive activities, such as anti-tumour, anti-bacterial, anti-viral, anti-inflammatory, immunomodulatory, hypotensive, hypoglycaemic, hypolipidaemic, anti-oxidant, free radical scavenging, and hepatoprotective effects [13][14][15][16][17][18][19][20][21][22][23][13,14,15,16,17,18,19,20,21,22,23]. More importantly, its excellent qualities and remarkable efficacy have also been highlighted in clinical applications, which are mainly used for the treatment of cancer, hypertension, diabetes, pelvic inflammatory disease, thyroid, and prostrate disorders [24].
E. ferox is widely distributed throughout tropical and subtropical regions of Asia and Southeast Asia. India, Japan, Korea, Bangladesh, and China are the main producing areas [6]. Generally, E. ferox is grown in stagnant water with a depth of 0.2–2.0 m, such as ponds and lakes (Figure 1A). It prefers warm and sunny weather and is intolerant to cold and drought. The suitable temperature ranges from 20 to 30 °C, while fertile soil with sufficient organic matter is required [6][25][6,25]. The plant bears 8–9 spherical leaves, while bright purple flowers are arranged in alternating rows and interlaced similar to an octopus. The flower is epigynous with more than 40 corollas, ovary 7–16 chambered with 6–8 seeds/locule. The seeds are spherical with a diameter of 0.5–0.8 cm, the surface is covered with a brownish-red or reddish-brown inner seed coat, one end is yellow-white, accounting for about 1/3 of the whole, and there is concave seed cleft hilum. The roots are long, fleshy, fibrous, usually in 2–3 clusters with numerous stomata (Figure 1C) [7]. The whole plant of E. ferox is edible except for the leaves [6]. In China, E. ferox is divided into southern and northern varieties. Northern E. ferox, also known as prickly E. ferox, is a wild species with purple flowers. It is mainly distributed in Hongze Lake and Baoying Lake of Jiangsu province (Figure 1E). Southern E. ferox, also known as Su Qian, is a variety of northern E. ferox after artificial domestication and cultivation, with larger leaves and white, red, or purple flowers (Figure 1D). Nevertheless, the southern E. ferox is mainly used for food and export, while the northern E. ferox is for medicinal applications [1][4][1,4].
The National Research Center for Makhana, Darbhanga (ICAR) estimates that the total area of E. ferox cultivation in India is 15,000 ha, while in China, Shangrao, Jiangxi Province is the biggest cultivation city with 6700 ha every year. In India, 120,000 metric tonnes of E. ferox seeds were produced each year, the produce is valued at Rs 250 crore at the farmer’s end. India exports about 1–2% of its overall output and almost 100 tonnes of popped E. ferox are shipped each year [10]. It is worth noting that the growing and processing of E. ferox is laborious and demanding work. To maintain plant-to-plant spacing, thinning seedlings are required after planting, while seeds collected from the pond are needed during harvesting. Subsequently, processing including storage, cleaning, grading, heating, and tempering is required, all of which are performed manually. The whole operation is therefore strenuous and painful work from planting to harvest.

2. 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][26][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 [27][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][28][29][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 H2O2-induced injury on HUVEC and VSMC through enhancement of T-AOC, SOD, and CAT activities and decrease of MDA content [30][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.

3. 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 [31][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 streptozotocin-induced Wistar rats over a four-week period. After 45 days of gavage consisting of 2β-hydroxybetulinic acid 3β-caprylate (HBAC) and 2β-hydroxybetulinic acid 3β-oleiate (HBAO) in diabetic mice, the plasma glucose and insulin were normalized, pancreatic β-cell, the histological architecture of pancreas, kidney, and liver were restored, as well as the endogenous antioxidant enzymes [33][34][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 IC50 of EFSSE on α-amylase and α-glucosidase inhibitory effect was 62.95 and 52.06 μg/mL, respectively [35][57]. The hypoglycemic and hypolipidemic effects of triterpenoid-rich 75% ethanol extracts of E. ferox shell were investigated in streptozotocin-induced 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 [36][53]. Triterpenoid-rich 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 [37][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 [38][60].
Currently, network pharmacology as an emerging discipline has been gradually applied to the mechanistic study of phytopharmaceuticals. This method is suitable for multi-component 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 [39][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.

4. 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 post-ischemic ventricular function and reduced myocardial infarct size in a chronic ischemic reperfusion model. Two cardioprotective proteins, TRP32, and thioredoxin, were significantly increased.

5. 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 [40][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 [41][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 glutamate-induced 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 [43][62].

6. 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.

7. 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.

8. 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 [44][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 [45][64]. In addition, the methanolic E. ferox seed and leaf extracts showed anti-fungal effects against Candida albicans and Pencillium notatum strains [46][65].