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

Abstract

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.

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]. 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]. 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].

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]. 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]. 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]. 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].

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.

Although the main constituents and bioactivities of EFS have been extensively investigated, detailed summaries are few. Furthermore, the pharmacological mechanisms of active components remain to be reviewed. Thus, we aim to perform a comprehensive, in-depth, and systematic review of EFS’s traditional applications, phytochemistry, pharmacological effects, and toxicity over the past 30 years. This review will provide quotable evidence for future studies on EFS and contribute to shedding further light on the biological activities and clinical applications of EFS.

2. Methodology

To retrieve information relevant to this review, an extensive search of the literature was conducted using multiple databases, including Google Scholar, Web of Science, PubMed, SciFinder, China National Knowledge Infrastructure (CNKI), and the China Science and Technology Journal database. The main keywords searched were “E. ferox”, “Foxnut”, “Lotus seeds”, “Gorgon nuts”, or “Phool Makhana”. Subsequently, the keywords “chemical composition”, “phytoconstituents/phytochemicals”, “biological activity”, “pharmacological activity”, and “toxicology” were used to refine the publications. E. ferox-related articles were retrieved from peer-reviewed journals worldwide ranging from 1980 to 2023. These articles were read thoroughly during the compilation and integration of the information to evaluate the authenticity and relevance of their information. Studies lacking scientific names were excluded, as well as studies on the uses of EFS in fields other than the medical and nutritional sciences. All chemical structures were validated by Sci Finder and drawn using Chem Draw Ultra 15.0.

3. Traditional Medicinal Uses of E. ferox

The unique natural conditions and long-term practice in different regions have led to versatile lifestyles and unique experiences in the treatment of certain diseases. EFS is generally used as an economic crop in India, Japan, Korea, Bangladesh, and other Southeast Asian countries [26]. In China, EFS was first recorded in “Shen Nong’s Classic of the Materia Medica” and described as “sweet and astringent tastes, and used for dampness and paralysis, pain in the lumbar spine and knees, tonifying and removing malignant diseases, benefiting the essence, strengthening the will, and making the ears and eyes wise” [5], and most of the subsequent records followed this statement with modifications. Current Chinese Pharmacopoeia (2020 Edition) recorded EFS as “sweet, astringent, and flat, attributes to the spleen and kidney meridians, benefiting the kidney and consolidating sperm, tonifying the spleen and inhibiting diarrhoea, eliminating dampness and arresting leucorrhea. It is used for spermatorrhea, enuresis and frequent urination, splenoasthenic diarrhea, and leucorrhea” [9]. In India, EFS is known as makhana and is applied in Ayurvedic medicines for treating diseases including bile disorders, persistent diarrhea, kidney disorders, rheumatic disorders, excessive leucorrhea, hepatic dysfunctioning, etc. [27]. While in Japan, EFS is recorded in Kampo medicine for improving metabolic arthritis, urinary incontinence, and leucorrhea. In addition to EFS, the whole plant of E. ferox can be used as food or medicine. As early as the report in the Compendium of Materia Medica in the Ming dynasty, China, the stems, leaves, and roots of E. ferox were applied to treat different diseases. To ascertain the efficacy and traditional uses of E. ferox, we summarized the records in ancient herbal works or research reports (Table 1).

Table 1.

The traditional uses of E. ferox.

Parts	Herbal Records	Dynasty/Country	Effects	Ref.
Seeds	Shen Nong’s Classic of the Materia Medica (Shén Nóng Bĕn Căo Jīng, 神农本草经)	Eastern Han Dynasty, AD 25–220, China	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 Bǎi 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 Cǎo 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]

4. 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 Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8 and Figure 9.

Figure 2.

Chemical structures of polyphenols and flavonoids of E. ferox.

Figure 3.

Chemical structures of cyclic peptides of E. ferox.

Figure 4.

Chemical structures of cerebrosides of E. ferox.

Figure 5.

Chemical structures of steroids and pentacyclic triterpenes of E. ferox.

Figure 6.

Chemical structures of tocopherols of E. ferox.

Figure 7.

Chemical structures of lignans of E. ferox.

Figure 8.

Chemical structures of essential oils of E. ferox.

Figure 9.

Chemical structures of other compounds of E. ferox.

4.1. 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].

4.2. 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 ultrasonic-assisted 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 (9–11). 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.

Table 2.

Phytochemical compounds identified from E. ferox.

No.	Compounds	Molecular Formula	Type	Plant Part	Analytical Methods	Ref.
1	EPJ	-	Polysaccharide	Seeds	CC, FT-IR	[31]
2	EFSP-1	-	Polysaccharide	Seeds	GPC, FT-IR, GC-MS, NMR	[32]
3	Gallic acid	C7H6O5	Polyphenol	Seed shells, Seeds	TLC, MS, NMR, HPLC	[12,16,36]
4	Pyrogallol	C6H6O3	Polyphenol	Seed shells	HPLC	[36]
5	Chlorogenic acid	C16H18O9	Polyphenol	Seed shells	HPLC	[36]
6	Resveratrol	C14H12O3	Polyphenol	Seeds	HPLC	[16]
7	4-O-methyl gallic acid	C8H8O5	Polyphenol	Seeds	NMR, MS, HPLC	[37]
8	Protocatechuic acid	C7H6O4	Polyphenol	Seeds	HPLC	[12]
9	Naringenin	C15H12O5	Flavonoid	Seed shells, Seeds	HPLC, GPC, LC-ESI-MS/MS, NMR, FT-IR, UV, IR, HR-EIMS	[18,38,39]
10	Dihydrotricetin	C15H12O7	Flavonoid	Seed shells	HPLC, LC-ESI-MS/MS	[18]
11	Pectolinarigenin	C17H16O6	Flavonoid	Seeds	HPLC	[12]
12	Rutin	C27H30O16	Flavonoid	Seed shells	HPLC	[36]
13	Cyclo(Pro-Ser)	C8H12O3N2	Cyclodipeptide	Seeds	GPC, NMR, FT-IR, MS	[38]
14	Cyclo(Ile-Ala)	C9H16O2N2	Cyclodipeptide	Seeds	GPC, NMR, FT-IR, MS	[38]
15	Cyclo(Leu-Ala)	C9H16O2N2	Cyclodipeptide	Seeds	GPC, NMR, FT-IR, MS	[38]
16	Cyclo(Phe-Ser)	C12H14O3N2	Cyclodipeptide	Seeds	GPC, NMR, TR, MS	[40]
17	Cyclo(Ala-Pro)	C8H12O2N2	Cyclodipeptide	Seeds	GPC, NMR, TR, MS	[40]
18	Cyclo(Phe-Ala)	C12H14O2N2	Cyclodipeptide	Seeds	GPC, NMR, TR, MS	[40]
19	N-α-hydroxy-cis-octadecaenoyl-l-O-β-glucopyranosylsphingosine	C42H79O9N	Cerebroside	Rhizomes with adventitious root	CC, EI-MS, NMR	[41]
20	Peracetylated cerebroside	C54H91O15N	Cerebroside	Rhizomes with adventitious root	CC, EI-MS, NMR	[41]
21	(2S,3R,4E,8E,2′R)-1-O-(β-glucopyranosyl)-N-(2′-hydroxydocosanoyl)-4,8-sphingadienine	C44H83O9N	Cerebroside	Seeds	CC, MPLC, IR, UV, HRFAB-MS, EI-MS, NMR	[42]
22	(2S,3R,4E,8E,2′R)-1-O-(β-glucopyranosyl)-N-(2′-hydroxytetracosanoyl)-4,8-sphingadienine	C46H87O9N	Cerebroside	Seeds	CC, MPLC, IR, UV, HRFAB-MS, EI-MS, NMR	[42]
23	β-sitosterol	C29H50O	Steroid	Seeds	HPLC	[12]
24	Daucosterol	C35H60O6	Steroid	Seeds	HPLC	[12]
25	Fucosterol	C29H48O	Steroid	Seeds	HPLC, NMR, MS	[37]
26	24-methylcholest-5-enyl-3β-O-pyranoglucoside	C43H49O10	Steroid	Rhizomes with adventitious root	TLC, CC, NMR, MS	[43]
27	24-ethylcholest-5-enyl-3β-O-pyranoglucoside	C44H51O10	Steroid	Rhizomes with adventitious root	TLC, CC, NMR, MS	[43]
28	24-ethylcholesta-5,22E-dienyl-3β-O-pyranoglucoside	C44H49O10	Steroid	Rhizomes with adventitious root	TLC, CC, NMR, MS	[43]
29	2β-hydroxybetulinic acid 3β-caprylate	C38H62O5	Pentacyclic triterpene	Seeds	HPLC, NMR, MS, FT-IR, UV	[44]
30	2β-hydroxybetulinic acid 3β-oleiate	C48H80O5	Pentacyclic triterpene	Seeds	CC, FT-IR, ESI-MS, NMR, UV	[45]
31	α-tocopherol	C29H50O2	Tocopherol	Seeds	GPC, TLC, NMR, FT-IR, MS, GC-MS, HPLC, UV	[38,46,47]
32	β-tocopherol	C28H48O2	Tocopherol	Seeds	GPC, TLC, NMR, FT-IR, MS	[38]
33	δ-tocopherol	C27H46O2	Tocopherol	Seeds	GPC, TLC, NMR, FT-IR, MS	[38]
34	Ferotocotrimer C/E	C86H142O6	Tocopherol	Seeds	CC, TLC, MPLC, IR, UV, HRFAB-MS, NMR, HR-ESI-MS, CD	[42,48]
35	Ferotocotrimer D	C86H142O6	Tocopherol	Seeds	CC, TLC, MPLC, IR, UV, HRFAB-MS, NMR, HR-ESI-MS, CD	[42]
36	Tocopherol trimer IVa	C87H144O6	Tocopherol	Seeds	CC, TLC, MPLC, IR, UV, HRFAB-MS, NMR, HR-ESI-MS, CD	[42]
37	Tocopherol trimer IVb	C87H144O6	Tocopherol	Seeds	CC, TLC, MPLC, IR, UV, HRFAB-MS, NMR, HR-ESI-MS, CD	[42]
38	Ferotocodimer A	C58H98O5	Tocopherol	Seeds	CC, TLC, MPLC, IR, UV, HRFAB-MS, NMR, HR-ESI-MS, CD	[48]
39	Euryalin A	C31H38O10	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
40	Euryalin B	C30H36O9	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
41	Euryalin C	C32H40O12	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
42	rel-(2R,3β)-7-O-methylcedrusin	C20H24O6	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
43	syringylglycerol-8-O-4-(sinapyl alcohol) ether	C23H30O9	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
44	(1R,2R,5R,6S)2-(3,4-dimethoxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0]octane	C20H22O6	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
45	(+)-syringaresinol	C22H26O8	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
46	Buddlenol E	C31H36O11	Lignan	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[18,39]
47	(+)-Isolariciresinol 9’-O-glucoside	C26H34O11	Lignan	Seeds	GPC, TLC, NMR, FT-IR, MS	[38]
48	3-(4-hydroxy-3-methoxybenzyl)-4-[(7’R),5’-dihydroxy-3’-methoxybenzyl] tetrahydrofuran	C20H24O6	Lignan	Seeds	HPLC, NMR, MS	[37]
49	Furfural	C5H4O2	Volatile constituents	Seeds	GC-MS	[46]
50	Pentanoic acid	C5H10O2	Volatile constituents	Seeds	GC-MS	[46]
51	2-methyl-3-pentanone	C6H12O	Volatile constituents	Seeds	GC-MS	[46]
52	5-methyl-2-furancarboxaldehyde	C7H14O	Volatile constituents	Seeds	GC-MS	[46]
53	Hexanoic acid	C6H12O2	Volatile constituents	Seeds	GC-MS	[46]
54	4, 4, 8-trimethyl-non-7-en-2-one	C12H22O	Volatile constituents	Seeds	GC-MS	[46]
55	1-(2-butoxyethoxy)-2-propanol	C9H20O3	Volatile constituents	Seeds	GC-MS	[46]
56	Phenol	C6H6O	Volatile constituents	Seeds	GC-MS	[46]
57	2-methylphenol	C7H8O	Volatile constituents	Seeds	GC-MS	[46]
58	4-methylphenol	C7H8O2	Volatile constituents	Seeds	GC-MS	[46]
59	4-ethylphenol	C8H10O	Volatile constituents	Seeds	GC-MS	[46]
60	Isocreosol		Volatile constituents	Seeds	GC-MS	[46]
61	4-ethylguaiacol	C9H12O2	Volatile constituents	Seeds	GC-MS	[46]
62	2, 6-dimethoxyphenol	C8H10O3	Volatile constituents	Seeds	GC-MS	[46]
63	4-methoxy-2, 3, 6-trimethylphenol	C10H14O2	Volatile constituents	Seeds	GC-MS	[46]
64	3, 4-dimethoxytoluene	C9H12O2	Volatile constituents	Seeds	GC-MS	[46]
65	3-tert-butyl-4-hydroxyanisole	C11H16O2	Volatile constituents	Seeds	GC-MS	[46]
66	1, 2, 3-trimethoxybenzene	C9H12O3	Volatile constituents	Seeds	GC-MS	[46]
67	[3.1.1] hept-3-en-2-one, 4, 6, 6-trimethyl-bicyclo	C10H14O	Volatile constituents	Seeds	GC-MS	[46]
68	Butylated hydroxytoluene	C15H24O	Volatile constituents	Seeds	GC-MS	[46]
69	1S, 4R, 7R, 11R-1, 3, 4, 7-tetramethyltricyclo [5.3.1.0(4, 11)] undec-2-en-8-one	C15H22O	Volatile constituents	Seeds	GC-MS	[46]
70	2, 6-bis (1, 1-dimethylethyl)-2, 5-cyclohexadiene-1, 4-dione	C14H20O2	Volatile constituents	Seeds	GC-MS	[46]
71	2-methylnaphthalene	C₁₁H₁₀	Volatile constituents	Seeds	GC-MS	[46]
72	Pentamethyl benzene	C11H16	Volatile constituents	Seeds	GC-MS	[46]
73	1-ethylidene-1H-indene	C11H10	Volatile constituents	Seeds	GC-MS	[46]
74	Tridecane	C13H28	Volatile constituents	Seeds	GC-MS	[46]
75	Pentadecane	C15H32	Volatile constituents	Seeds	GC-MS	[46]
76	Hexadecane	C16H34	Volatile constituents	Seeds	GC-MS	[46]
77	Dodecane	C12H26	Volatile constituents	Seeds	GC-MS	[46]
78	Tetradecane	C14H30	Volatile constituents	Seeds	GC-MS	[46]
79	Heptadecane	C17H36	Volatile constituents	Seeds	GC-MS	[46]
80	Octadecane	C18H38	Volatile constituents	Seeds	GC-MS	[46]
81	Nonadecane	C19H40	Volatile constituents	Seeds	GC-MS	[46]
82	Palmitic acid	C16H32O2	Volatile constituents	Seeds	GC-MS	[46]
83	Linoleic acid	C18H32O2	Volatile constituents	Seeds	GC-MS	[46]
84	Ethyl gallate	C9 H10O5	Ester	Seeds	HPLC	[12]
85	4-hydroxybenzylethyl ether	C8H10O2	Ether	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
86	5,7-dihydroxychromone	C9H6O4	Ketone	Seeds	HPLC	[12]
87	ω-hydroxypropioguaiacone	C10H12O4	Ketone	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
88	Coniferyl aldehyde	C10H10O3	Aldehyde	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
89	Trans-p-hydroxycinnamaldehyde	C9H8O2	Aldehyde	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
90	p-hydroxybenzaldehyde	C7H6O2	Aldehyde	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
91	p-hydroxybenzyl alcohol	C7H8O2	Alcohol	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
92	p-hydroxyphenethyl alcohol	C8H10O2	Alcohol	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
93	2-methoxybenzene-1,3-diol	C7H8O3	Phenyl alcohol	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
94	4-ethoxyphenol	C8H10O2	Phenol	Seeds	CC, HPLC, NMR, UV, IR, HR-EIMS, ESIMS	[39]
95	Resorcinol	C6H6O2	Phenol	Seeds	HPLC, NMR, MS	[37]
96	Alliin	C6H11NO3S	Sulfoxide	Seeds	HPLC	[16]
97	Adenosine	C10H13N5O4	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
98	Guanosine	C10H13N5O5	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
99	Cytidine	C9H13N3O5	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
100	Uridine	C9H12N2O6	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
101	Inosine	C10H12N4O5	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
102	Thymidine	C10H14N2O5	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
103	2′-deoxyadenosine	C10H13N5O3	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
104	2′-deoxyguanosine	C10H13N5O4	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
105	2′-deoxycytidine	C9H13N3O4	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
106	2′-deoxyuridine	C9H12N2O5	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
107	2′-deoxyinosine	C10H12N4O4	Nucleoside	Seeds	HPLC-ESI-TQ-MS/MS	[49]
108	Xanthine	C5H4N4O2	Nucleobase	Seeds	HPLC-ESI-TQ-MS/MS	[49]
109	Hypoxanthine	C5H4N4O	Nucleobase	Seeds	HPLC-ESI-TQ-MS/MS	[49]
110	Thymine	C5H6N2O2	Nucleobase	Seeds	HPLC-ESI-TQ-MS/MS	[49]
111	Adenine	C5H5N5	Nucleobase	Seeds	HPLC-ESI-TQ-MS/MS	[49]
112	Cytosine	C4H5N3O	Nucleobase	Seeds	HPLC-ESI-TQ-MS/MS	[49]

4.3. 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 (13–18) have been isolated from EFS. The isolated cyclic peptides are shown in Table 2 and Figure 3.

4.4. 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.

4.5. Steroids and Pentacyclic Triterpenes

Steryl glycosides (SGs) and acylated steryl glycosides (ASGs) are two major derivatives of sterols. Steroidal glycolipids are considered to be unique glycolipids that play an important role in the structure of cells. Three sterols, β-sitosterol (23), daucosterol (24), and fucosterol (25) were isolated from EFS ethyl acetate extract [12,37]. While, three glucosylsterols named 24-methylcholest-5-enyl-3β-O-pyranoglucoside (26), 24-ethylcholest-5-enyl-3β-O-pyranoglucoside (27), and 24-ethylcholesta-5,22E-dienyl-3β-O-pyranoglucoside (28) were obtained from the rhizomes with adventitious roots of E. ferox [43].

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 3β-caprylate (30), from the ethyl acetate extract [44,45]. The specific information and structures of each compound are shown in Table 2, Figure 5.

4.6. 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 and purified with silica gel and Sephadex LH-20, Li et al. identified three tocopherols, α-tocopherol (31), β-tocopherol (32), and δ-tocopherol (33), from EFS [38]. In a search for bioactive constituents of EFS, Rowet al. identified two new tocopherol trimers, ferotocotrimer C (34) and D (35), and two known tocopherol trimers, IVb (36) and IVa (37) from EFS (16.1 kg) methanol refluxed extracts, their structures were determined on the basis of spectroscopic data, especially 1D and 2D NMR experiments [42]. In addition, two new tocopherol polymers, the chroman-type dimer ferotocodimer A (38) and the spiro-type trimer ferotocotrimer E (34), were isolated from EFS [48] (Figure 6). Their structures were determined based on spectroscopic data, especially 1D and 2D NMR analysis. The absolute configuration of 34 and 38 were determined by CD exciton chirality and ROESY experiment.

4.7. Lignans

Song et al. extracted EFS (50 kg) with 85% EtOH three times, the extract (1.2 kg) was suspended in water, and was successively partition with EtOAc, the components in the EtOAc extract of EFS (55 g) were investigated, 3 new sesquineolignans, named euryalins A-C (39–41), 2 neolignans, named rel-(2α,3β)-7-O-methylcedrusin (42) and syringylglycerol-8-O-4-(sinapyl alcohol) ether (43), and 3 furofuran-type lignans, named (1R,2R,5R,6S)2-(3,4-dimethoxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0]octane (44), (+)-syringaresinol (45), and buddlenol E (46), were identified. The relative configuration was determined by the ROESY while the absolute configuration remained undetermined [39]. In addition, another 2 antioxidative lignans, (+)-Isolariciresinol 9′-O-glucoside (47) and 3-(4-hydroxy-3-methoxybenzyl)-4-[(7′R),5′-dihydroxy-3′-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.

4.8. 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.

4.9. Others

Other phytochemicals reported in E. ferox include esters (84, 85), ketones (86, 87), aldehydes (88–90), alcohols (91, 92), phenols (93, 94, 95), and sulfoxide (96). In addition, 16 nucleosides and nucleobases (97–112) were simultaneously determined by HPLC-ESI-TQ-MS/MS, and their contents of them in 26 batch samples were quantified with standards [49] (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].

5. 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.

Table 3.

Pharmacological activities of E. ferox-derived extracts and phytoconstituents.

Activity	Compound/Extract	Plant Part	Animals/Cell Lines	Doses	Effects	Ref.
Antioxidation and anti-inflammation	Methanol extract	Seeds	DPPH scavenging assays V79-4 cell line RAW 264.7 cell line	For antioxidation, 0.8–100 μg/mL For antiinflammation, 100–400 μg/mL	DPPH radical scavenging activity (IC50 22.95 ± 0.25 μg/mL or 5.6 μg/mL); iNOS, Cox-2, NO inhibition (300–400 μg/mL); Inhibition of lipid peroxidation (IC50 20.5 μg/mL)	[19,20]
	Ethanol extract Compounds 40, 42–46	Seeds	DPPH scavenging assays ROS model: glucose-treated mesangial cells	For DPPH: 2–1000 μg/mL For ROS: 1, 10 μM	DPPH (SC50) of ethanol extract, compounds 40–43, 45 were 103.1 μg/mL, 6.8, 10.4, 10.2, and 12.9 μM, respectively; Compounds 40, 42, 44–46 showed ROS inhibition at 10 μM	[39]
	Aqueous extracts	Seeds	DPPH and ABTS scavenging activity H2O2-induced human skin fibroblast oxidative stress	/	DPPH and ABTS scavenging; Increased expression of SOD, CAT, and GSH-Px	[21]
	Phenolic extracts	Seed shells	DPPH scavenging assays	0.1–1.0 mg/mL	DPPH scavenging activity is similar to vitamin C and Trolox at 1 mg/mL	[36]
	Phenolic extracts	Seed shells	DPPH and Hydroxyl scavenging assays D-galactose-induced aging Kunming mice	In vitro: 0.01–2 mg/mL In vivo: 100, 200, 400 mg/kg p.o. once daily for 32 days	Strong DPPH and Hydroxyl scavenging activity; Increases the SOD, CAT, and GSH-Px activities, and decreases MDA content in the liver and kidneys	[18]
	Anthocyanins extraction	Leaves	DPPH and ABTS scavenging activity	/	DPPH and ABTS scavenging IC50 were 74.00 ± 3.63 μg/mL and 5.77 ± 0.28 μg/mL, respectively	[26]
	Ethyl acetate, ethanol, or 50% ethanol extract	Seed shells	DPPH scavenging assays Lipid peroxidation	5–200 μg/mL	DPPH radical scavenging activity (IC50 29.4 ± 1.34, 28.3 ± 1.21, 27.60 ± 1.02 μg/mL, respectively); Inhibition of lipid peroxidation (IC50 43.86 ± 1.32, 30.44 ± 1.15, 36.42 ± 1.43, respectively)	[54]
	Cell wall polysaccharides	Petioles and pedicels	DPPH and ABTS scavenging activity H2O2-induced injury on HUVEC and VSMC	For DPPH and ABTS: up to 6.5 mg/mL For cell model: 60 and 200 μg/mL	Around 80% DPPH scavenging activity at 3.25 mg/mL, and 100% ABTS scavenging activity at 1.625 mg/mL; Reduced MDA levels, and increased T-AOC, SOD, and CAT activities in H2O2-injured VSMC, and HUVEC cells.	[55]
	Essential Oil	Seeds	DPPH and ABTS scavenging activity	0.5, 1, 2, 4, and 8 μg/mL	DPPH and ABTS scavenging IC50 were 6.27 ± 0.31 and 2.19 ± 0.61 μg/mL, respectively	[46]
Antidiabetic and hypoglycemic effects	70% ethanol extract	Seeds	Streptozotocin-induced diabetic Wistar rats	100, 200, 300, 400 mg/kg, p.o. for 45 days	Significantly decreased the blood glucose level, increased plasma insulin level, restored hepatic gluconeogenic enzymes activities; increased activities of SOD, CAT, GPx, and GSH	[15]
	70% methanol extract	Germinated seeds	Streptozotocin-induced diabetic ICR mice	100, 200, 400 mg/kg, p.o. for 4 weeks	Improved hyperglycemia, abnormal lipid metabolism, and renal tissue lesions; Decreased kidney microalbuminuria, blood urea nitrogen, serum creatinine, MDA, and GSH; Increased activity of CAT, SOD, serum total antioxidant capacity; Regulating the Keap1/Nrf2/HO-1 and AMPK/mTOR pathways.	[56]
	Polysaccharide (EFSP-1)	Seeds	Dexamethasone-induced HepG2 and 3T3-L1 preadipocyte cells	Incubation with 25, 100, 400 μg/mL EFSP-1 for 24 h	Increasing glucose consumption by up-regulating the expression of GLUT-4 via activating the PI3K/Akt signal pathway in insulin resistance cells	[32]
	2β-hydroxybetulinic acid 3β-caprylate (HBAC)	Seeds	Streptozotocin-induced diabetic Wistar rats	20, 40, 60 mg/kg, p.o. for 45 days	Exhibited free radical scavenging property, pancreas, and hepatoprotective effect; Stimulating insulin release; Improved the glycemic control and lipid profile	[44]
	2β-hydroxybetulinic acid 3β-oleiate (HBAO)	Seeds	Streptozotocin-induced diabetic Wistar rats	20, 40, 60 mg/kg, p.o. for 45 days	Alleviating glycemic homeostasis and oxidative stress, normalized plasma glucose, glycosylated hemoglobin (HbA1c), hepatic gluconeogenic enzymes, plasma insulin, ameliorating pancreatic β-cell, hepatic and renal histology and β-cell functions, improving dyslipidemia and antioxidant enzymes	[45]
	Ethanol extract	Seed shells	α-amylase and α-glucosidase	20–100 μg/mL	The inhibitory effects of E. ferox seed shell extract (EFSSE) on α-amylase and α-glucosidase in terms of IC50 were 62.95 and 52.06 μg/mL, respectively.	[57]
	Triterpenoid-rich 75% ethanol extracts	Seed shells	Streptozotocin-induced diabetic mice	200, 400, 600 mg/kg, p.o. for 4 weeks	Regulating glucose metabolism and body weight; Decreased cholesterol, LDL, and triglycerides levels, and increased HDL	[53]
	Triterpenoid-rich 75% ethanol extracts	Seed shells	Streptozotocin-induced diabetic Kunming mice	200, 300, 400, 500 ± 2 mg/L in drinking water for 4 weeks	Restored glucose metabolism and body weight; Recovered Islet morphology; Reduced PTP1B protein and increased insulin receptor IRS-1 protein	[58]
	Network pharmacology method	/	The TCMSP, SymMap V2, CTD, DisGeNET, and GeneCards databases were searched for ES components, targets, and DKD targets	/	The main components are oleic acid and vitamin E, targeting the proteins PPARA, LPL, FABP1, and MAPK1 to regulate TNF, apoptosis, and MAPK.	[59]
	Polysaccharides	Petioles and pedicels	Alloxan-induced hyperglycemic ICR mice	100, 200, 400 mg/kg, p.o. for 28 days	High doses of EFPP reverse alloxan-induced body weight loss, reduce blood glucose level, enhance serum insulin level, improve oral glucose tolerance, increase hepatic glycogen content and GCK activity; increase SOD, CAT, and GSH-Px activities and decrease MDA contents in liver and kidney	[60]
Hepatoprotective and cardioprotective activities	Ethanol extract	Seed shells	High-fat diet-induced ICR mice	15 and 30 mg/kg, p.o. for 28 days	Reduced body weight, lipids deposition in the liver and blood lipids, decreased MDA content, and increased SOD activity; IRs-1 activation and CYP2E1 inhibition	[13]
	Ethanol extract	Seeds	Ischemia and reperfusion in vitro model; Chronic ischemic reperfusion injury in vivo model	25, 125, or 250 μg/mL for in vitro; 50 and 500 mg/kg, p.o. for 21 days	Improved post-ischemic ventricular function and reduced myocardial infarct size; increased expression of TRP32 and thioredoxin proteins; ROS scavenging activities	[14]
Anticancer	Ethanol extract	Seeds	In vitro: A549 Human Caucasian Lung Carcinoma cancer cells In vivo: Balb/c nu/nu mice	In vitro: 50–150 μg/mL In vivo: 100 mg/kg/day for 28 days	In vitro: promoting A549 apoptosis via inhibition of the Akt protein and activation of the p53 protein; In vivo: activating p53 and suppressing the tumor growth	[16]
	Ethanol extract	Seed shells	Human Gastric Cancer SGC7901 cells and Human Hepatoma HepG2 cells	50–800 μg/mL	Inhibitory effect on the proliferation of SGC7901 cells and HepG2 cells were 92.63% and 72.40%, respectively	[61]
	Ethyl acetate fraction	Seeds	Melan-a cells	3–30 μg/mL	Inhibition of cellular tyrosinase and melanin synthesis	[23]
	Resorcinol	Seeds	B16F10 melanoma cells	/	Inhibition of melanin synthesis in B16F10 melanoma cells with an IC50 492.8 µM	[37]
Cytotoxicity	Ferocerebrosides A and B	Seeds	Brine shrimp lethality bioassay	62.5, 125, 250, 500, and 1000 μg/mL for 24 h	Ferocerebrosides A and B showed marginal toxicity against brine shrimp with LC50 values of 0.17 and 0.20 mM, respectively	[42]
	Polysaccharide fraction (EFSP-1)	Seeds	3T3-L1 preadipocyte cells and HepG2 cells	25, 50, 100, 200, 400 μg/mL for 48 h	No obvious influence on cells at 100–400 μg/mL	[32]
	Hexane, diethyl ether, ethyl acetate extract	Seeds	Glutamate-induced cytotoxicity in hybridoma N18RE-105 cells	10 μg/mL for 24 h	Dose-dependent protection against neuronal cell death induced by 20 mM glutamate	[62]
Anti-fatigue	Phenolics extract	Seed shells	Exhaustive swimming test	100, 200, 400 mg/kg p.o. once daily for 32 days	The average exhaustive swimming time was obviously prolonged in all three doses	[18]
Anti-depressant	Petroleum ether fraction	Seeds	Chronically unpredictable mild stress (CUMS) mouse model	0.1, 0.15 g/kg p.o. once daily for 14 days	Upregulation of AMPK and ULK1, attenuated depressive behavior via AMPK-ULK1 pathway-mediated autophagy	[22]

Figure 10.

The summarized pharmacological mechanism and effects of E. ferox. Up arrow symbol means upregulation and down arrow symbol means downregulation.

5.1. 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].

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 [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.

5.2. 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 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 [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 [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 [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 [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 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 [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.

5.3. 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. 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].

5.4. 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 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 [62].

5.5. 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.

5.6. 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.

5.7. 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].

6. 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.

7. 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 expected 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 in-depth 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.

Author Contributions

Conceptualization, Z.Y. and J.J.; methodology, J.J.; software, Z.Y. and J.J.; investigation, J.J., H.O., H.L. and R.C.; writing—original draft preparation, J.J.; writing—review and editing, Z.Y. and L.Z.; supervision, Z.Y.; funding acquisition, Z.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not available.

Funding Statement

This research was funded by the Natural Science Foundation of Guangdong Province of China (grant number 2022A1515011419), Key Project in Higher Education of Guangdong, China (grant number 2022ZDZX2029), Scientific Research Foundation of Guangdong Ocean University (grant number R19035), and the Innovative Team Program of High Education of Guangdong Province (2021KCXTD021).