Diplazium esculentum (Retz.) Sw.: Ethnomedicinal, Phytochemical, and Pharmacological Overview of the Himalayan Ferns

The genus Diplazium (family: Athyriaceae) comprises approximately 350 species of pteridophytes. Diplazium esculentum (Retz.) Sw. is an important member of this genus and commonly known as a wild vegetable in the Himalayan and sub-Himalayan communities. According to the literature analysis, D. esculentum was traditionally used for the prevention or treatment of several diseases such as diabetes, smallpox, asthma, diarrhea, rheumatism, dysentery, headache, fever, wounds, pain, measles, hypertension, constipation, oligospermia, bone fracture, and glandular swellings. Various extracts of D. esculentum were evaluated to elucidate their phytochemical and pharmacological activities. A wide array of pharmacological properties such as antioxidant, antimicrobial, antidiabetic, immunomodulatory, CNS stimulant, and antianaphylactic activities have been recognized in different parts of D. esculentum. The review covers a systematic examination of pharmacognosy, phytochemistry, and pharmacological applications of D. esculentum, but scientifically, it is not fully assessed regarding complete therapeutic effects, toxicity, and safety in the human body. The published literature on D. esculentum and its therapeutic properties were collected from different search engines including Wiley online, PubMed, Springer Link, Scopus, Science Direct, Web of Science, Google Scholar, and ACS publications by using specific terms such as “Diplazium esculentum, bioactive compounds, biological activities and health benefits” from 1984 to 2021 (March). Therefore, further studies are required to identify the detailed action mechanism of D. esculentum in vitro/in vivo, and also, more studies should focus on conservation, cultivation, and sustainable utilization of the species.


Introduction
The Himalayan botanicals are well known to produce wide variety of secondary metabolites due to critical climatic conditions [1][2][3][4]. These botanicals, including wild plants, have a significant role in food security and socio-economic development of the region [5,6]. Moreover, these botanicals are locally utilized for food resources, medicines, and other purposes due to the presence of numerous bioactive compounds and high nutritional value [7,8]. With recent developments in science and technology, the importance of wild plants has been identified as a possible source of nutraceuticals and/or functional foods [9].
Among several high valued functional foods, Diplazium esculentum is one of the important species of wild ferns, which is frequently consumed by people living in the hilly areas; it is not growing on much higher altitude. D. esculentum (n = 41 chromosomes, grade of polyploidy = diploid) is utilized as a traditional vegetable in the Himalayan communities [10]. It is an important member of the genus Diplazium which comprises around~350 species of pteridophytes, mainly distributed in Asia and Oceania [11].
Recently, a few researchers have validated its nutraceutical and pharmacological properties by using in vitro and in vivo models/methods. For the development of evidencebased medicine, a critical investigation of current knowledge is required regarding ethnopharmacology, chemical composition, biological activities, and possible side effects of the species. Additionally, D. esculentum belongs to the least concern category under International Union for Conservation of Nature (IUCN) 2021-1 (https://www.iucnredlist.org/ species/194150/8883499) and needs more attention. Therefore, in this manuscript, we reviewed and discuss the recent scientific information conducted so far on D. esculentum, which includes its pharmacognosy, phytochemistry, and pharmacology.

Pharmacognosy
2.1. Traditional Uses. Traditionally, D. esculentum is one of the most popular vegetables consumed in different parts of the globe, namely, India, Philippines, Nepal, China, Thailand, Indonesia, etc. The literature has revealed that D. esculentum is still being used by different communities for the treatment of several diseases including diabetes, smallpox, asthma, diarrhea, rheumatism, dysentery, headache, fever, wounds, pain, measles, and high blood pressure. The detailed information on the traditional uses of the species is summarized in Table 1. Additionally, this species is collected in large amounts and marketed by the rural and tribal communities of India for their livelihood enhancement [22]. The Mishing community of Assam (State of the Indian republic) used it essentially in the religious ceremony of the dead person [23].

Proximate and Mineral
Composition. The nutritional value of any food substance can be analyzed by its proximate and mineral composition [24]. Literature-based screening of the proximate composition of D. esculentum revealed the presence of lipids, proteins, carbohydrates, vitamins, fiber, etc., while mineral composition possesses the presence of essential micro and macro compounds [25][26][27][28][29][30][31][32][33][34][35]. The comparative analyses of proximate and mineral composition of D. esculentum are presented in Tables 2 and 3.

Biological Applications
Among the functional properties of D. esculentum, the antioxidant, anti-inflammatory, antimicrobial, antidiabetic, and immune-modulatory activities can be considered as potentially contributing to the preventive and pharmacological values of this plant species (Figure 4). The following sections reviewed the abovementioned functional biological activities of different D. esculentum extracts.

Antioxidant Activities.
The botanicals can be considered as safe and cost-effective natural antioxidants capturing free radicals and may help in the prevention and the treatment of different diseases [79,80]. Recently, a research group from Indonesia reported that the methanolic extract of D. esculentum showed a good antioxidant activity with an IC 50 value of 123.95 ppm according to 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay [2].
In another study, the antioxidant activity of D. esculentum, extracted by using pressurized hot water extraction (PHWE) method, was reported [81]. The results demonstrated that the optimum condition for the best antioxidant activity of PHWE was at 175°C, 21 min extraction time (2 g dried powder in 50 mL water) in Box-Behnken design. The plant extract showed moderate DPPH scavenging activity (EC 50 = 1241:14 μg/mL). The hydro-alcoholic extract of D. esculentum leaf was evaluated for antioxidant activity using the DPPH and nitric oxide assays [82]. The IC 50 value of the plant extract for DPPH and NO inhibition activity was found to be 138.8 and 151.9 mg/mL, respectively.

Antimicrobial Activities.
Recently, several pathogenic microorganisms have developed antibiotic resistance, and these antibiotics can have undesirable side effects [92]. Thus, researchers are focusing on botanicals for the development of herbal-based antibiotic substitutes [93]. Table 5 includes antimicrobial studies performed with D. esculentum. Antimicrobial activity was considered good (minimum inhibitory concentration (MIC) less than 100 μg/mL), moderate (MIC from 100 to 500 μg/mL), weak (MIC from 500 to 1000 μg/mL), or inactive (MIC over 1000 μg/mL). Inactive results of antimicrobial activities of D. esculentum did not included in this study [46,94].
Different parts (leaves, rhizomes, and roots) of the D. esculentum were extracted with aqueous and alcoholic sol-vents to evaluate the antibacterial activity by using the disk diffusion method. Four bacterial strains, namely, E. coli, Salmonella arizonae, Salmonella typhi, and Staphylococcus aureus, were used in this study. The rhizome and root extracts inhibited the growth of microorganisms while leaf extract did not show any inhibition. Additionally, extracts  [39,40] Hairs are removed, boiled with salt and water until water is evaporated then fried and eaten as vegetable. India [41][42][43][44][45] Used in headache, pain, fever, wounds, dysentery, glandular swellings, diarrhea, measles, toothache, high blood pressure, and various skin infections. Fronds used by pregnant women as protection against difficult childbirth. Leaf paste is used in the wounded place externally for the cure of bone fracture. Used as a laxative. Used as insecticides.
Bangladesh, Nigeria, Indonesia, Nigeria, India, Philippines [15-17, 19, 46-48] The tender frond is cooked without salt and is consumed with rice for 5-10 days for the treatment of diabetes. India [20] Eaten as highly preferred Koche Sag, Neuro/Niuro vegetable. Nepal [49] Used as vegetable and pickle. India, Vietnam, Japan, Indonesia, Philippines [30,31,[50][51][52][53] Root About 20 g of fresh root is boiled in 1 liter of water and reduced to onefourth of its volume. 3 mL of this decoction along with 2 mL of honey is taken orally on an empty stomach twice a day for 15 days to cure spermatorrhea.
India [54] About 50 g juice obtained from macerated root is fed three times for human dysentery. Macerated root extract is also useful for the cattle dysentery.
Bangladesh [55] About 2-3 spoonsful of root juice are taken for 1/2 days, or 1/½ cup of boiling extract of whole plant is taken thrice daily to treat infections and used as an antidote. The root paste is used externally for the treatment of rheumatism and smallpox. Two pills of pulverized root and honey are taken thrice daily for 2 weeks for the treatment of oligospermia.

Oxidative Medicine and Cellular Longevity
Trichophyton mentagrophytes. The plant extract only showed a good-moderate antimicrobial activity against I. orientalis [97].
The methanolic extract of D. esculentum leaves has been evaluated for antibacterial activity by using the disc diffusion method [98]. The plant extract showed slight antibacterial activity (6-10 mm zone of inhibition) against Salmonella paratyphi, Vibrio parahaemolyticus, E. coli, B. megaterium, Shigella dysenteriae, and Shigella boydii among 12 bacterial strains.
The chloroform and methanolic extracts of D. esculentum leaves were evaluated for antimicrobial activity by using the disk diffusion method [46]. The plant extracts showed inactive antimicrobial activity against all the microbial strains tested, namely, K. pneumoniae, S. aureus, E. coli, Salmonella typhimurium, Vibrio cholerae, Sarcina lutea, The antifungal activity of D. esculentum leaves against three fungal strains using the agar diffusion method has been reported [94]. The methanolic extract showed inactive antifungal activity against Aspergillus niger, Rhizopus stolonifer, and C. albicans in terms of MIC (50-100 mg/mL) and minimum fungal inhibition concentration (100-200 mg/mL).

Antidiabetic Activities.
Diabetes mellitus is a chronic carbohydrate, fat, and protein metabolism disorder characterized by the increase in blood glucose level due to defect of insulin secretion [99]. The inhibition of α-glucosidase and α-amylase enzymes, involved in the digestion of carbohydrates, can significantly reduce the postprandial increase of blood glucose and therefore can be an important strategy in the management of blood glucose level in type 2 diabetic and borderline patients. The antidiabetic activity of D. esculentum through inhibition of α-glucosidase and α-amylase enzymes has been reported [69]. The results demonstrated that D. esculentum extract exhibited the highest α-amylase (92.09%) and α-glucosidase (70.01%) inhibitory activities.
The protective effect of a hydro-alcoholic extract of D. esculentum on streptozocin-(STZ-) induced diabetes was evaluated [82]. In this study, a total of 30 rats were used and treated with plant extract up to 21 days. After the treatment, it was observed that the plant extract (500 mg/kg) reduced (50.2%) the blood glucose level in STZ-induced diabetic rats. Additionally, a significant reduction was recorded in plant extract-treated rats for lipid profiling (p < 0:01), serum marker enzyme activity (p < 0:001), necrosis, and regeneration of beta cells. The plant extract showed dosedependent activity in all the experiments. 4.4. Immunomodulatory Activity. The immunosuppressive and hemolytic activities of D. esculentum extracts in mouse models have been evaluated [100]. A total of 120 Swiss albino mice (6-8 weeks age) were treated with plant extracts up to 180 days. After this treatment, the plant extract showed significant dose-dependent decreases in body weight, relative spleen weight, number of plaques (formation of antibody secreting cells) formed, hemagglutination antibody titer value, the number of peritoneal macrophages, and the number of cultured splenocytes. The in vitro analysis showed significant dose-dependent increases in the percentage inhibition of splenocyte proliferation as well as the percentage of hemolysis. In other words, the treatment with D. esculentum may act as an immunosuppressive agent.
The impact of boiled D. esculentum on Th1 and Th2 cytokine levels of Swiss albino mice that were treated with different doses of plant extract, daily up to 180 days, has been reported [101]. The outcome of the study demonstrated that the plant extract significantly decreases the concentration of Th1 and Th2 cytokines when compared with controls. In other words, boiled D. esculentum extract may affect some of the innate and cell-mediated immune responses by modulating the level of Th1 and Th2 cytokines.     [102]. In this cross-sectional study, a total of 132 adults were recruited. Socio-demographic information, anthropometric measurements, dietary history, food frequency, and cognitive function were assessed. The average ulam intake by the participants was 15:1 ± 8:2 g/day. The outcome of the study indicated that "pucukpaku" showed protective effects (62.9%) against cognitive decline. The anticholinesterase and NADH oxidase inhibitory activities of a methanolic extract of D. esculentum have been evaluated [83]. Recently, most of the studies reported that the inhibition of anticholinesterase has been shown to be a strategy for the treatment of neurodegenerative disorders. The results of the study demonstrated that the methanolic extract of D. esculentum inhibited acetyl-cholinesterase and NADH oxidase in a dose-dependent manner, with IC 50 values of 272.97 and 265.81 μg/mL.
The CNS stimulant effect of D. esculentum in a mouse model using digital acto-photometer has been reported [88]. The plant water extract showed statistically significant (p < 0:0001) and dose-dependent activity when compared with control and standard caffeine.
4.6. Toxicity Studies (In Vitro and In Vivo). The methanolic and chloroform extracts of D. esculentum were evaluated for their toxicity using brine shrimp lethality bioassay. Both extracts produced dose-dependent increment in percent mortality of brine shrimp nauplii which indicates the presence of toxic compounds in the extracts. The LC 50 values were recorded as 1.87 μg/mL (chloroform), 1.62 μg/mL (methanol), and 0.66 μg/mL (vincristine sulphate as standard drug) [46]. In another study, the toxicity of methanolic extract of D. esculentum using brine shrimp lethality bioassay was reported as significant (LC 50 = 18:6 μg/mL). [98]. In other study, the cytotoxicity of ethanolic extract of D. esculentum was evaluated in different cell lines including breast cancer (MDA-MB-231 and MCF-7), colon cancer (Caco-2), liver cancer (HepG2), and normal liver (Chang liver), and no cytotoxic effect was observed [103].
The systemic toxicity and several pathological effects of D. esculentum were evaluated on rabbits and guinea pigs [104]. The study indicated that the plant extract decreased all the pathological functions including growth, body weight, forced motor activity, alterations of blood glucose values, erythrocyte sedimentation rate, mean corpuscular volume, mean corpuscular hemoglobin, total leukocyte count, neutrophil, lymphocyte, and monocyte count, while increased blood SGOT level in both rats and guinea pigs. In other words, the plant extract indicated toxic effects in guinea pigs and rabbits, while rats showed a little adverse effects. Junejo and coworkers reported the nontoxic effects of D. esculentum extract on experimental models and recommended as a potential functional food [67].
The toxicological impact of D. esculentum on male reproductive functions of Swiss albino mice has been reported [105]. A total of 120 male Swiss albino mice of 6-8 weeks of age were fed orally with 80, 160, and 320 mg/kg b.w. of plant material and treated up to 180 days. After this successful treatment, the boiled plant extract showed significant dose and time-dependent decreases in body weight, absolute and relative testis    [108]. The plant extracts combined with polyaluminium chloride showed a synergistic effect for all the measured parameters in Kuala Sepetang Landfill Site (KSLS) leachates. The combination was identified as a high molecular weight polymer, and it acted as an anionic coagulant and was also capable of promoting the coagulation process. Extracts of the rhizome of D. esculentum extracts were evaluated for their anthelmintic activity against Pheretima posthuma. The study included three solvents (ethanol, aqueous, and petroleum ether) and three concentrations (10, 25, and 50 mg/mL), and all the extracts demonstrated significant anthelmintic activity in terms of the time of paralysis and time of death. Ethanolic extract showed the highest activity compared to other solvents, and the activity was recorded in dose-dependent patterns [109].
Silver nanoparticles (10-45 nm) were synthesized using the leaf powder of D. esculentum [110]. The synthesized nanoparticles were evaluated as a catalyst in the degradation of methylene blue and rhodamine B and also evaluated for the anticoagulation activity. The synthesized Ag NPs showed considerable anticoagulation activity. Besides, prominent photocatalytic activity in the degradation of methylene blue and rhodamine B was also recorded.
The antitrypanosomal activity of D. esculentum leaves was evaluated against Trypanosoma brucei brucei strain BS221 [111]. In this study, the ethanolic extract was used with seven different concentrations (0.01 to 12.5 μg/mL), and the extract showed significant antitrypanosomal activity with IC 50 value 4.32 μg/mL and a selectivity index (SI) value > 23 in mammalian cell line (Vero, IC 50 > 100 μg/mL) when compared with the positive control (pentamidine, I C 50 = 4:51 ng/mL).

Concluding Remark and Future Prospective
The present manuscript reports traditional uses, nutraceuticals, pharmacognosy, phytochemistry, and pharmacological studies in D. esculentum. The literature survey revealed that D. esculentum is one of the most important and popular wild species of ferns in the Himalaya. It is a widely used species in different traditional systems, but the complete chemical composition and active compounds need to be further elucidated and authenticated by bioassay-guided isolation. However, very limited studies are available for this species, not only in terms of chemical characterization but also in terms of pharmacological evaluation as well. Most of the studies are limited to the in vitro screening and a few for in vivo. Clinical trial studies should be performed to evaluate the safety profile of wild ferns in the human body in terms of antimicrobial activity, antidiabetic activity, antiinflammatory activity, and immunomodulatory aspects. Apart from this, educating the local people regarding the cultivation, conservation, and sustainable utilization of this plant will help for improving the population size of the species.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declared that they have no conflict of interest regarding this manuscript. Oxidative Medicine and Cellular Longevity