Abstract: Background and Objectives: Ferns are among very useful but neglected, threatened and underutilized group of plants in Nigeria. The aim of the study was to document and highlight the ethnobotanical values and mode of germination of these fern tubers for utilization and conservation. Materials and Methods: The phytochemical constituents and proximate compositions of the tubers of two species of Nephrolepis were investigated using standard methods. Germination and growth pattern of potted tubers of both species were also monitored for two consecutive growing seasons. Results: Both tubers were found to contain alkaloids, cardiac glycosides, flavonoids, saponins, triterpenes while steroids were absent solely in N. cordifolia. From proximate analysis, moisture (75%), carbohydrate (12%) and dry matter (24%) contents were almost in same proportion; protein [N. undulata (3.6%), N. cordifolia (1.3%)], while the ash content was the least [N. cordifolia (1%), N. undulata (0.5%)]. Germination of tubers commenced in N. cordifolia at 1 month and 5 months in N. undulata, while tubers got matured within 4 months. Conclusion: The study showed that these tubers contained nutrients and phytochemicals that are nutritionally important for human health and as feeds for livestocks.
INTRODUCTION
Ferns and lycophytes are significant components of the rainforest ecosystem which serve as good ecological indicators1. The genus Nephrolepis belongs to the family Nephrolepidaceae in the Eupolypod 1 Clade2. Nephrolepis is pantropical in distribution, consisting of approximately 30 species3. Oloyede and Odu4 reported six species of Nephrolepis in southwestern Nigeria. Nephrolepis are epiphytic, lithophytic or terrestrial ferns species that occur abundantly in Nigeria. Rhizomes are short creeping or erect tuberous and with numerous stolons. Fronds are pinnate, articulated and monomorphic with hydathodes close to the margin in some species and veins are free, rachises often persistent. Sori are reniform (kidney shaped) with scale-like indusium. According to Nwosu5, the tubers of N. cordifolia is boiled with salt and eaten in the eastern part of Nigeria while the fronds and rhizomes are used as amnesia. Oloyede et al.6 elucidated the useful phytochemical constituents in 6 Nephrolepis species, such as tannins, saponins and flavonoids. The antimicrobial properties of frond extracts of Nephrolepis bisserata and Psilotum nudum were reported in India7. Agbodeka et al.8 reported that fronds of N. undulata were among plants used to treat malaria in Plateau region of Togo. The extracts of rhizomes of Nephrolepis cordifolia have been used in India as contraceptive as well9. Apart from ethnobotanical uses, Nephrolepis species are widely used as ornamental plants. Some uses of N. cordifolia including the tubers have been reported, but tubers of N. undulata have not gained ethnobotanical recognition up till now.
Hence, the aim of this study was to investigate the tuber propagation and determine the phytochemical constituents and proximate compositions of the two Nephrolepis species in order to explore their possible propagation and utilization as food to humans and livestock.
MATERIALS AND METHODS
Matured tubers of N. cordifolia and N. undulata were harvested from the Museum Fern Garden of Obafemi Awolowo University, Ile-Ife, Nigeria. Tubers were sown separately in 15 kg pots of soil at the rate of one tuber per pot in five replicates. Planting was done in mid-August, 2017 within the Fern Garden (N 07°31ʹ9.73; E 004°31ʹ35.9). Adequate watering was carried out every other day. Emergence and growth were monitored and recorded monthly. Phytochemical analysis of tubers were carried out using standard procedure as described by AOAC10.
Phytochemical screening
Test for alkaloids: To test for alkaloids, 0.5 g of the extract was added to 5 mL of 1% aqueous hydrochloric acid and stirred on a steam bath and then filtered. Then 1 mL of the filtrate was mixed with few drops of Mayer’s reagent and to another 1 mL of the filtrate was supplemented with Dragendorff’s reagent. Precipitation or turbidity of these reagents indicated the presence of alkaloids.
Test for saponins: To test for saponins, 0.5 g of the extract was vigorously shaken in a test tube; persistence of frothing on warming indicated the presence of saponins.
Test for flavonoids: To test for flavonoids, 0.5 g of the extract was added to few drops of 1% Aluminium chloride. The presence of yellow precipitate indicated the presence of flavonoids.
Test for cardiac glycosides: To determine the presence of cardiac glycoside, 0.5 g of the extract was dissolved in 2 mL of chloroform to which sulphuric acid was carefully added to form a lower layer. A reddish brown coloration at the interphase indicated the presence of steroidal rings.
Test for triterpenes: About 20 mg of the extract was suspended in 10 mL of chloroform, warmed slightly in water bath and filtered. Afterwards, the chloroform layer was added to small volume of sulphuric acid which was properly stirred, the appearance of color indicated the presence of triterpenes.
Test for steroids: For this purpose, 0.5 mL of concentrated sulphuric acid was added to 1.0 mL of the extract and was allowed to stand for 5 min. Reddish brown precipitate indicated the presence of steroid.
Proximate analysis
Moisture content: Moisture content was determined by drying 3.0 g of the samples in a hot oven, Uniscope (SM9053) at 105°± until a constant weight was observed. The sample was removed from the oven and the result obtained was expressed as percentage (%) of the dry matter.
Protein: The total protein content was determined using the Kjeldahl method. Ground sample (0.2 g) was weighed into a Kjeldahl flask. About 10 mL of concentrated sulphuric acid was added followed by one Kjeltec tablet (Kjeltec-Auto 1030 Analyzer, USA). The mixture was digested on heating racket to obtain a clear solution. The digestate was cooled, transferred onto Kjeldahl distillation, set up and 75 mL distilled water was added followed by 50 mL of 40% sodium hydroxide solution. The ammonia formed in the mixture was sub-sequently distilled into 25 mL, 2% boric acid solution containing 0.5 mL of the mixture of 100 mL of bromocresol green solution and 70 mL of methyl red solution indicators. The distillate collected was then titrated with 0.1 M HCl to determine the percentage nitrogen in a blank solution. The nitrogen content was multiplied by a conversion factor 6.25 to obtain crude protein content.
Ash content: Ash content was determined by the official AOAC11 method using muffle furnace (Carbolite AAF1100, United Kingdom). About 5 g (W3) of the sample was weighed into already weighed (W2) ashing crucible and placed in the muffle furnace chambers at 550°C until the samples turned into ashes usually within 5 h. The crucibles were removed, cooled in a desiccator and weighed (W1). Ash content was expressed as percentage of weight of the original sample as shown in following equation:
| W1 | = | Weight of crucible+ash |
| W2 | = | Weight of empty crucible |
| W3 | = | Weight of sample |
Crude fibre: The crude fibre was determined as described by AOAC11, 2 g (W3) of sample was digested with 200 mL of 1.25% sulphuric acid followed by 200 mL of 1.25% NaOH and washed with 50-70 mL of distilled water after each digestion, the solution were filtered, dried in an oven at 130°C, cooled and weighed (W1). The crucible was then combusted at 550°C in a muffle furnace chamber for 3 h, cooled and reweighed (W2). The crude fiber content as a percentage of the sample were calculated by the expression:
Carbohydrate: Carbohydrate was expressed as a percentage of the difference between the addition of other proximate chemical components and 100 as shown in below equation:
Carbohydrate (%) = 100 - (moisture content + ether extract + crude fibre + ash content + crude protein)
Statistical analysis: Proximate compositions were analyzed by standard error of means while simple percentage (%) was used to estimate germination of the tubers.
RESULTS
Phytochemical analysis: Phytochemicals detected from both cold and hot water extracts (Table 1) of the two studied tubers includes: alkaloids, cardiac glycosides, flavonoids, saponins, triterpenes while steroids were absent solely in N. cordifolia extracts.
Proximate analysis: The results in Table 2 revealed the proximate composition of that the studied tubers. Moisture (75.005%), carbohydrate (12.92%) and dry matter (24.995%) contents were almost in the same proportion; followed by protein [N. undulata (3.614%), N. cordifolia (1.318%)] while the ash content was the lowest [N. cordifolia (1.085%), N. undulata (0.57%)].
Growth pattern: As shown in Fig. 1, emergence of new fronds from the croziers in N. cordifolia commenced within one month (August) after planting while in N. undulata, about 6 months after planting. Germination percentage in Table 3 was very high in both fern tubers; 100% in N. cordifolia and 98% in N. undulata. Proliferation of leaflets from only one tuber was up to 16 and 10 in N. cordofolia and N. undulata, respectively after a period of 10 months (August-May). Tubers were ready to be harvested within 4 months after emergence in both species (Fig. 2).
| Table 1: | Phytochemical screening of the Nephrolepis tubers |
| +: Present, -: Absent | |
| Table 2: | Proximate analysis of Nephrolepis tubers |
| NC: Nephrolepis cordifolia, NU: Nephrolepis undulata | |
DISCUSSION
The presence of alkaloids, cardiac glycosides, flavonoids, saponins, triterpenes and steroids in N. undulata only confirmed that the tubers contained useful bioactive components. Pharmaceutical activities such as antimicrobial, antioxidant and anti-inflammatory have been reported in phytochemicals from ferns and lycophytes12-17. The proximate analysis showed comparable nutritional compositions especially in moisture content, carbohydrate and dry matter. The proportion of carbohydrate and proteins in N. cordifolia tubers were almost same with the reports of Gauchan et al.18 from Nepal in Asia. The variations observed in other nutrient components of the tubers may be due to environmental factors.
| Table 3: | Germination percentage and leaflet proliferation in two Nephrolepis |
| n = 5 | |
Fiber is useful in cleansing the digestive tracts by removing the potential carcinogens from the body thereby preventing the absorption of excess cholesterol19 the high quantity of starch and minimal protein and fiber highlights the nutritive values of the tubers.
| Fig. 1: | Growth patterns in Nephrolepis cordifolia and Nephrolepis undulata |
| Nc: Nephrolepis cordifolia, Nu: Nephrolepis undulata | |
| Fig. 2(a-d): | Plant forms, (a) Fronds of N. cordifolia showing tubers, (b) Tubers of N. cordifolia, (c) Fronds Nephrolepis undulata showing tubers and (d) Tubers of N. undulata |
The high moisture contents of the two tubers indicated that the harvested tubers cannot be stored for a long period; however, high moisture content in food sample is known to provide for greater activity of water soluble enzymes and co-enzymes needed for metabolic activities20. The experiment on the tuber propagation which showed that the tubers are good perennating structures for both plants. This may explain why the plants have persistent nature. Both terrestrial and epiphytic forms of N. undulata did not tolerate drought but with the underground tubers, new growth resumes immediately after rains. Similarly, the phenological reports on six Nephrolepis species showed that the croziers of N. undulata unfolded rapidly but had the shortest aerial life span than other Nephrolepis species including N. cordifolia21. It is noteworthy to report that in spite of watering, there was no germination in N. undulata until after two to three rains in February, at this same period which the epiphytic N. undulata forms started to emerge as evidenced by new croziers and this was continuous throughout the raining season. In this study and in earlier observations, N. cordifolia was relatively drought tolerant and showed steady growth continuously all year round. Frond proliferation was higher in N. cordifolia than in N. undulata. It has been established that the colonization and growth of epiphytic ferns to a large extent are strictly dictated by climatic factors22. This implied that terrestrial or aquatic ferns can grow through all year round once water is available. The inability of N. undulata to germinate in pots with watering until after few drops of rain seems unclear but may indicate the specific condition necessary for the germination of N. undulata. However, further investigations on anti-nutritional and phytotoxic profiles of the tubers are recommended to validate safety in consumption of the fern tubers.
CONCLUSION
This study showed useful phytochemicals and proximate compositions in the fern tubers highlighted their pharmaceutical and nutritive values while the rapid proliferation of leaflets connoted easy tuber production. The high germination percentage of tubers can serve as a promising field character in proposing the fern tubers especially N. cordifolia for large scale propagation.
SIGNIFICANCE STATEMENT
The study discovered that the two underutilized tubers are potential sources of nutrients and bioactive compounds that can be beneficial to human health status. The study will help the researchers to uncover critical areas involving cultivation of the tubers on a large scale. Thus a new theory on the usefulness of the tuber for food, as source of pharmaceutical compounds and large scale production for commercial purposes may be arrived at.