Diplazium esculentum (Family: Athyriaceae), one of the most popular wild edible fern, is considered as a pharmacologically diverse ethnomedicinal plant. The coiled fronds and young leaves of this plant have a wide range ethnomedicinal uses in folk medicine around the world directed for a number of ailments. The present article is the first comprehensive review on the pharmacological activities of this particular edible fern. The structural formulas of all the chemical constituents identified and isolated so far from D. esculentum are also provided in the present review. The most thoroughly studied pharmacological activities viz., antioxidant, antimicrobial, immunomodulatory, neuromodulatory, anti-fertility, cytotoxic, etc.) of the D. esculentum extracts have been discussed. This comprehensive review will be of help for the future researchers investigating for more potent compounds and their pharmacological activities.

Ferns are one of the most widely used wild edible groups of plants throughout the world. Their origin have been dated back to the Paleozoic era and they constitute the primitive vascular plant group which is found scattered all over the world. Due to their primitive origin, ferns have been well adapted with various changes of the environment than other primitive vascular plants of the world (Wallace et al. 1991). Therefore, ferns are expected to have many useful phytochemicals than other plants. The presence of these diverse groups of phytochemicals largely influence the pharmacological properties of ferns when they are consumed, which ultimately determines its beneficial and / or detrimental effects on human and animal health. It is interesting to note that only a few of the fern groups are used as food throughout the world. The fern stems, rhizomes, leaves, young fronds and shoots, and sometimes the whole plants are used for food (Liu et al., 2012). Diplazium esculentum (Koenig ex Retz.) Sw. (Family – Athyriaceae) is one such fern which is considered as one of the most commonly consumed edible ferns throughout the world.

Though there are some literatures on the ethnobotanical and ethnomedicinal studies of D. esculentum, the epidemiological studies of this fern have not yet been attempted. The studies done so far on this fern were mainly concerned with its beneficial effects either in vitro or in vivo in small laboratory animals. Very few researchers have focused on its health impacts. Most of the studies on this fern have been conducted to assess the antioxidant, antimicrobial, antitumor or other beneficial activities (Nanasombat & Teckchuen, 2009; Tongco et al., 2014; Kaushik et al., 2012; Seal, 2012) but very few studies have been performed so far to determine the possible pharmacological and toxicological impacts of this fern on human and animal health. In a nut shell, no comprehensive review of this plant has been reported to date which demonstrates all the bioactivities of this plant. Therefore, the overall aim of the present review is to elucidate the potential therapeutic prospects as well as health deteriorating properties of D. esculentum taking consideration on the latest and updated information on this plant, and thereby to advance the existing knowledge of this fern as food and medicine in relation to human health.

D. esculentum is an edible fern, pantropical in distribution and occurs widely and commonly throughout India, China, Cambodia, Laos, Thailand, Vietnam and Malaysia. It grows in gregarious colonies in open marshy areas, stream banks and canals from sea level to 2,300 m. The rhizome is erect, often forming a slender leaning black trunk to 1 m tall, scaly at the apex. Scales are 1 cm long, dark brown, margins finely toothed, apex long-acuminate. Fronds are 1–2 m long, 0.5–1 m wide, erect to arcuate. Stipe is black and scaly at the base, paler above. Lamina is 2–3-pinnate, 0.5–1.5 m long, 0.5–1 m wide, dark green. Secondary pinnae variable in size, commonly 5–8 cm long, 1.5–2.5 cm wide, margins very shallowly lobed, lobes are toothed, basal lobes longer than the rest, glabrous beneath, veins are simple or forked, lowest 3–5 pairs of adjacent vein groups anastomosing. Sori spreading along most veins; indusium thin, dark brown, margins becoming uneven with age (Roy, 2017).

Kingdom: Plantae

Division: Pteridophyta

Class: Polypodiopsida

Order: Polypodiales

Family: Athyriaceae

Genus: Diplazium

Species: D. esculentum

Figure 1.

Diplazium esculentum is one of the most common varieties and the most commonly consumed fern throughout Asia and Oceania. In India, young fronds of D. esculentum are popularly known as lingra in Northern India, rukja and lochanch in North Eastern India and dheki sak in West Bengal, India. The newly emerging coiled fronds are consumed after cooking as a seasonal vegetable during monsoon season which continues for almost five months. The frond of this fern is generally cooked in oil or butter; using them in a vegetable curry is less preferred (Roy et al., 2013a). In the northeastern India, especially in Sikkim, and in the central and north-western Himalayan states of India (Himachal Pradesh and Uttarakhand), the local folk relish both vegetables and pickles from D. esculentum. Natives consider these recipes effective both to counteract constipation and as an appetizer, especially as a pickle (FAO, 2010). Study conducted in the villages of the Parvati valley, Himachal, India revealed that out of the 50 consumed wild edibles, D. esculentum is used as a vegetable/pickle by an average of 66% of the inhabitants (Kala, 2005). In Malaysia, this plant is eaten as ‘ulam’ or green edible leaves, usually consumed with hot sauce. This practice of eating ‘ulam’ with sauce is also known as ‘krawoo’ (Rahmat et al., 2003).

The Apatani tribe of Arunachal Pradesh, India uses the frond of Diplazium esculentum plant as medicine for constipation (Kala, 2005). It has been shown that the local inhabitants of Similipal Biosphere Reserve, Orissa, India used to take honey with decoction of boiled water extract of D. esculentum in empty stomach twice a day for 15 days to cure spermatorrhea (Rout et al., 2009). The natives of Adi tribe of Dehang-Debang Biosphere Reserve of Arunachal Pradesh, India use the boiled young fronds of D. esculentum as vegetables with boiled rice for laxative purpose (Kagyung et al., 2010). In the Kolli hills of Eastern Ghats, Tamil Nadu, India, the natives use handful of D. esculentum leaves to make juice and taken orally twice a day to get relief from cold and cough (Karthik et al., 2011). Moreover, the local people of this region use the frond parts of this plant as laxative and often used to treat colitis and constipation (Perumal, 2010). The local people of Manokwari, West Papua Province use this plant for wound healing and as an ailment of headache (Lense, 2011). In a Sudanese community in Indonesia, D. esculentum is used for the treatment of fever, dermatitis and measles (Roosita et al., 2008).

6.1. Phytochemical analysis of D. esculentum

D. esculentum is reported to possess a diverse group of phytochemicals, viz., alkaloids, anthraquinones, anthranol glycosides, cardiac glycosides, cyanidins, flavonoids, glucosides, leucoanthocyanins, phenolic compounds, phytosterols, saponins, steroids, tannins and terpenoids. (Das et al., 2013, Tongco et al., 2014; Akter et al., 2014). The bioactive compounds present in this fern are esculentic acid, 5-O-Methyleriodictyol 7-O-(4-O-D-xylosyl)-β-D-galactoside, pterosin B, ptaquiloside, lutein, phosphoric acid, phytol, 2,6,10-trimethyl,14-ethylene-14-pentadecene, hexadecanoic acid methyl ester, pentadecanoic acid, Stigmasta-5,22-dien-3-ol, acetate, (3beta), beta-ocimene, 1,2- Benzenedicarboxylic acid, BIS(2-Methylpropyl)ester, 1-Heneicosanol, 5,8,11,14-eicosatetraenoic acid, methyl ester (all Z), ergost-5-en-3-ol, (3beta) and stigmast-5-en-3-ol, (3beta). (Tandon et al., 1980; Srivastava et al., 1981; Gangwar, 2004; Somvanshi, 2006; Wali et al., 2016; Naik et al., 2020). The chemical structures of these bioactive principles are depicted in Figure 2.

Figure 2.

Figure 3.

6.2. Antioxidant and free radical scavenging activities of D. esculentum 

The in vitro total antioxidant activity evaluated by ABTS, FTC, TBA, FRAP and phosphomolybdenum methods exhibit high percentages of the total antioxidant activity in D. esculentum which contains high amount of flavonoid and phenolic compounds that confers moderately high DPPH radical scavenging activity as evident by its IC50 value (Roy et al., 2013a; Das et al., 2013, Tongco et al., 2014; Wali et al., 2016). Oxidative stress of the liver was estimated by assessing the antioxidant enzymes catalase and reduced glutathione in Wistar rats. It was observed that the treatment of the rats with hydroalcoholic extract of D. esculentum significantly reduced the level of lipid peroxidation. The level of the antioxidant enzymes, i.e., catalase and reduced glutathione was also decreased significantly in a dose-dependent manner, indicating potent antioxidant potential of D. esculentum (Junejo et al., 2018). The methanolic extract of D. esculentum also possesses scavenging activities against different reactive oxygen species (ROS) and reactive nitrogen species (RNS), including hydroxyl, superoxide, hydrogen peroxide, singlet oxygen, hypochlorous acid, nitric oxide and peroxynitrite. Moreover, the D. esculentum extract acted as an iron chelator and also possessed reducing power and lipid peroxidation inhibition property. Moreover, D. esculentum is reported to contain significant amount of flavonoids and Phenolic compounds. Both these classes of compounds have good antioxidant potential and their effects on human nutrition and health are considerable (Roy et al., 2013a). 

6.3. Antibacterial activity of D. esculentum

The aqueous extracts of rhizome and root of D. esculentum inhibite the bacterial growth, whereas, the leaf extract did not show any inhibition. The aqueous and alcoholic extracts of rhizome were found to be more effective than the antibiotic. The synergistic effect was observed when the root extract was applied with the antibiotic against Salmonella arizonae and Staphylococcus aureus. But, the leaf and root extracts did not show any inhibition against E.coli. (Amit et al., 2011). The chloroform extract of D. esculentum showed strong antimicrobial activity against Sarcina lutea, Salmonella typhimurium, Bacillus subtilis, Klebsiella pneumonia, Shigella boydii, Escherichia coli, Staphylococcus aureus and Vibrio cholera. The highest zone of inhibition for chloroform extract was observed against Sarcina lutea followed by Salmonella typhimurium. On the other hand, in case of the aqueous extract, the highest zone of inhibition was found in Samonella typhimuriun followed by Klebsiella pneumonia (Akter et al., 2014).

6.4. Antidiabetic activity of D. esculentum

The antidiabetic activity of D. esculentum was reported by Junejo et al. (2018). They showed that D. esculentum significant lowered the blood glucose level in streptozotocin (STZ) induced diabetic rats. A 45.6

Phytochemicals are chemical compounds or chemical constituents formed in the plant's normal metabolic processes. The chemicals are often referred to as "secondary metabolites" of which there are several classes including alkaloids, steroids, terpenoids, catecholamines, tannins, saponins, anthraquinones, coumarins, fats, flavonoids, glycosides, gums, iridoids, mucilages etc. These naturally occurring phytochemicals are generally considered to be safe alternatives for synthetic drugs and found to be useful in the prevention of several diseases, and also have antimicrobial, antifungal, antiparasitic, antiviral, anti-allergic, antispasmodic, antihyperglycemic, anti-inflammatory, and immunomodulatory properties (Rabi & Bishayee, 2009; Wagner & Elmadfa, 2003; Sultana & Ata, 2008; Shah et al., 2009). The present study reviewed several promising pharmacological activities which are intrigued by extensive variety of potential phytoconstituents of D. esculentum.

One of the major groups of secondary metabolites present in D. esculentum is alkaloids which is reported to act on the nervous system as stimulators, exhibit acetylcholinesterase inhibition activity, enhance memory in animals and is also being investigated for the treatment of Alzheimer's disease (Zhang et al., 2002; Hirasawa et al., 2003, Ma & Gang, 2004). D. esculentum is also reported to possess flavonoids and Phenolic compounds. Among flavonoids, some exhibit neuroprotective activity against cytotoxic stress, suggesting their possible use in treatment of neurodegenerative diseases such as stroke and Alzheimer’s disease. The mechanism of action of flavonoids is through scavenging or chelation (Cook & Samman, 1996). Phenolic compounds are also very important plant constituents because their hydroxyl groups confer scavenging ability (Diplock, 1997).

D. esculentum is also investigated for potential bioactive principles among which esculentic acid and lutein are important. Esculentic acid is a triterpene which showed potent anti-inflammatory activity in mice. It is reported to inhibit pro-inflammatory cytokines like TNF-α and IL-6, inflammatory mediators viz., NO & PGE-2 in macrophages, and decrease the COX-2 protein expression in mice (Niu et al., 2014). On the other hand, lutein, an oxygenated carotenoid, acts as an antioxidant, protecting cells against the damaging effects of free radicals. It is hypothesized that they protect against visual disorders and cognition diseases, such as age-related macular degeneration (AMD), age-related cataract (ARC), cognition diseases, ischemic/hypoxia induced retinopathy, light damage of the retina, retinitis pigmentosa, retinal detachment, uveitis and diabetic retinopathy (Jia et al., 2017). Dietary odd-chain saturated fatty acids (OCFAs) like pentadecanoic acid and heptadecanoic acid are associated with lower risks of cardiometabolic diseases, and their higher dietary intake is associated with lower mortality (Venn-Watson et al., 2020). On the other hand, hexadecanoic acid methyl ester shows a variety of pharmacological activities viz., antioxidant, antifungal, hypocholesterolemic, antimicrobial, pesticide, and nematicide activities (Mustapha et al., 2016; Arora et al., 2017). Other beneficial bioactive constituents like 1-heneicosanol and β-ocimene possess antibacterial and antifungal, antitumor, and pest resistance activities, whereas, phytol possesses anxiolytic, metabolism-modulating, cytotoxic, antioxidant, autophagy- and apoptosis-inducing, antinociceptive, anti-inflammatory, immunomodulating, and antimicrobial effects (Arancibia et al., 2016; Russo & Marcu, 2017; Islam et al., 2018). 

Apart from these beneficial phytochemicals, D. esculentum possesses some toxic compounds as well, among which saponins and tannins are pharmacologically most important. Study revealed that both the crude and boiled D. esculentum possess hemolytic activity which is due to the presence of considerable amount of saponins in plant. Saponins have the capacity to destroy cell membrane, therefore may be related to the hemolytic potential. On the other hand, tannins inhibit protein availability through denaturation. Tannins are heat resistant compounds that can withstand high temperature during boiling. Thus, the toxic effects shown by D. esculentum could be attributed to tannins and other heat stable compounds (Roy et al., 2013b). Among the bioactive principles, ptaquilosides and pterosin B are the most prominent in this fern. D. esculentum collected from the high-altitude area of Harsil-Gangotri (Northern India) had 19 mg/kg Ptaquiloside (Somvanshi et al., 2006). Shade- and freeze dried samples of D. esculentum showed the presence of 10.94 to 16.36 mg/kg of pterosin B (Gangwar, 2004). During metabolism, ptaquiloside undergoes a series of reactions and produces a reactive aglycone dienone intermediate, the inactive pterosin B and DNA adducts. Ptaquiloside is activated at alkaline pH, which is considered as the reason for the location of tumors in the urinary bladder of ruminants and the ileum of rats (Smith et al., 1994).

Among other bioactive phytoconstituents, pthalic acid is the most important. Phthalic acid is already reported to present in considerable amount in D. esculentum. Phthalates are the diesters of phthalic acid which was reported to reduce the ovarian weight and increased uterine weight in female mice. In utero or lactational exposure of pthalates alters steroidogenic gene expression in the ovary, decreases estrogen synthesis, reduces ovarian weight, and reduces ovulation. Exposure of phthalates during fetal development altered follicular recruitment and development, eventually causing premature ovarian failure. Low molecular weight phthalates are also suspected of acting as hormones and cause infertility, overweight, and diabetes in men (Lyche, 2017; Barakat & Ko, 2018; Henkel, 2018).

One of the most important properties of D. esculentum investigated so far is the acetylcholinesterase inhibitory activity, which can explain the neuroimmune function of this plant. Acetylcholine (ACh) is a ubiquitous neurotransmitter and found even in the roundworm Caenorhabditis elegans, one of the simplest organisms with a nervous system (Rakowski et al., 2013; Kosinski & Zaremba, 2007). The wider significance of ACh is in understanding the biological effects of tested toxins and/or medical drugs: as any immunological effects of Acetylcholinesterase (AChE) inhibitors can involve both CNS and PNS, this has to be taken into consideration in interpreting any findings (Pohanka, 2014). The cholinergic system is tightly associated with the cholinergic anti-inflammatory pathway dominantly located in blood and mucosa. This pathway is a regulatory link between nerve terminations in blood and macrophages expressing the α7 nicotinic acetylcholine receptor (α7 nAChR) on their surface (Pohanka, 2012; Wessler & Kirkpatrick, 2008; Rosas-Ballina & Tracey, 2009). Discovery of the cholinergic anti-inflammatory pathway allows us to understand how the CNS is involved in the regulation of innate immunity (Pohanka, 2014). Acetylcholinesterase (AChE) bound on erythrocytes plays an important role in termination of cholinergic anti-inflammatory pathway activation (Pohanka, 2012; Silva-Herdade & Saldanha, 2013). The bioactive constituents like lutein and phytol, present in D. esculentum can inhibit AChE, and thus aid in the continued progression of the cholinergic anti-inflammatory pathway. The detailed mechanism of D. esculentum mediated regulation of the cholinergic anti-inflammatory pathway is illustrated in the Figure 3.

Figure - 3: The modulation of the cholinergic anti-inflammatory pathway via protection of ACh from splitting by cholinesterase and thus enhancing the pathway. Acetylcholine (ACh) is released by β2-adrenergic receptor T cells coupled with choline acetyltransferase (β2-AR T Cells + ChAT) following the stimulation of splenic nerve and subsequent release of norepinephrine, which binds β2-AR cells stimulating ACh release. At this point, the enzyme, acetylcholinesterase (AChE), which is bound to the erythrocytes, comes in to play and it cleaves the ACh molecule in to acetic acid and choline group, terminating the stimulation of the cholinergic anti-inflammatory pathway (Cuoco et al., 2016). The bioactive principles of D. esculentum (lutein, phytol, etc.) inhibit the AChE, and therefore, the circulating ACh molecules bind to macrophages via the α7nAChR activating IκB or JAK/STAT pathway to inactivate the NF-κB-mediated transcription of pro-inflammatory cytokines, such as IL-1, IL-6, TNF-α and CRP.

Diplazium esculentum have survived from the Paleozoic times and have adapted with many more various changes of environment than the other primitive vascular plants (Wallace et al., 1991). Therefore, this plant is expected to have many useful phytochemicals than other plants. It is interesting to note that not all the ferns are edible, only a few of them are used as food throughout the world, and D. esculentum is one of them. It is the most commonly consumed fern in the world. The fern stems, rhizomes, leaves, young fronds and shoots, and sometimes the whole plants are used for food (Liu et al., 2012). In recent years, more and more researches have reported the food and ethnomedicinal uses of this fern in different parts of the world but very few studies have been conducted so far to assess the pharmacological or toxicological impact of this plant on human health. This plant is enormous source of a wide range of compounds having diverse pharmacological and medicinal properties. The present review is the first attempt to summarize all the available information regarding the chemical profiles and pharmacological properties of this edible plant which may serve as the baseline data to do extensive studies towards the discovery of more potent compounds and further investigations for their biological activities. Therefore, further research should be carried out on D. esculentum to uncover the unidentified compounds, their functions including synergistic effects and mechanisms of action. Experimental studies including clinical trials are highly required to establish disease-specific optimal dosing and the optimal method to deliver the therapeutic agent.