Evaluation of Glucosidase Inhibitory and Cytotoxic Potential of Five Selected Edible and Medicinal Ferns

Purpose: To evaluate the glucosidase inhibitory and cytotoxic activities of five selected edible and medicinal ferns, namely, Blechnum orientale, Davallia denticulata, Diplazium esculentum, Nephrolepis biserrata


INTRODUCTION
Worldwide, many fern species are used as traditional remedies for human diseases and also consumed as vegetables.Ferns are rich in natural products with therapeutically-relevant bioactivities, including anti-cancer, antioxidant, and anti-inflammatory activities [1].Hence, ferns are promising bioresources for the discovery of bioactive compounds that can be exploited for the development of nutraceutical, cosmetic, and pharmaceutical products [1,2].

Phytochemicals
with cytotoxic and antiglucosidase activities can be used in the development of chemopreventive and antidiabetic therapies [12,13].Alpha (α)-glucosidase is one of the key therapeutic targets in the management of type 2 diabetes mellitus.Hence, antiglucosidase natural products may be incorporated into nutraceuticals and functional food, and in turn, used in the management of diabetes [1,2,14].The antiglucosidase activities of the five ferns have not been previously investigated.Likewise, nothing is known about the cytotoxic or anticancer potential of N. biserrata and P. vittata.Information on the anticancer effects of the other three ferns is also limited.
The health-promoting and therapeutic effects of the various classes of phenolic compounds derived from ferns and other plants have been established [1,14].However, information on the polyphenol, hydroxycinnamic acid, flavonoid, and proanthocyanidin contents of the five selected ferns is limited.Hence, to fill in gaps of knowledge about the bioactivities of these ferns and to identify promising fern species for isolation of active compounds in future, this study had two objectives: first, to assess the antiglucosidase and cytotoxic activities of the aqueous extracts of the five ferns; second, to determine whether such bioactivities can be attributed to the phenolic, hydroxycinnamic acid, flavonoid, and proanthocyanidin contents of the five selected ferns.

Preparation of aqueous extracts
The fern samples were oven-dried at 45 o C for 72 h and then pulverised to powder with a Waring blender.Aqueous extracts were prepared by suspending the powder in autoclaved, deionised water at a ratio of 1:20 (dry weight: volume).The mixture was heated in a 90 o C water bath for 1 h.The extract was vacuumfiltered and the resulting filtrate was centrifuged at 7830 rpm at 4 o C for 5 min.The supernatant obtained was freeze-dried to constant weight and extract yield was recorded.The freeze-dried extract was then redissolved in deionised water to prepare aliquots of 50 mg/ml, which were then stored at -20 o C until further use.

Determination of α-glucosidase inhibitory and cytotoxic activities
α-glucosidase inhibitory activity was assessed as previously described [15].A reaction mixture containing 250 μL of 100 mM potassium phosphate buffer (pH 7.0), 150 μL of 0.5 mM 4nitrophenyl α-D-glucopyranoside, 50 μL of extract, and 150 μL of α-glucosidase (0.1 unit/mL in 10 mM potassium phosphate buffer, pH 7.0) was incubated at 37 °C for 30 min.The reaction was terminated by adding 600 μL of 200 mM Na 2 CO 3 .The absorbance of the reaction mixture was recorded at 400 nm.A blank was prepared for each measurement by substituting αglucosidase with 10 mM potassium phosphate buffer.Antiglucosidase activity (Ag) was calculated according to Eq 1.
where As is the absorbance of control reaction (without extract) and Ac is the absorbance in the presence of an extract.Myricetin was used as the positive control.The effectiveness of myricetin as an α-glucosidase inhibitor has been established [16].Half maximal effective concentration (EC 50 ) value, defined as the concentration of extract or myricetin required to achieve 50 % antiglucosidase activity, was determined using linear regression analysis.
Cytotoxicity of the extracts against the human chronic myelogenous leukaemia cell line (K562) was assessed by using a methylthiazol tetrazolium (MTT) assay as previously described [17].5-Fluorouracil, an anticancer drug, was used as the positive control.EC 50 value, defined as the concentration of extract or 5-fluorouracil required for achieving 50 % cytotoxic activities, was determined by using linear regression analysis.

Phytochemical contents
Total phenolic (TP) content of the extracts was determined by using a Folin-Ciocalteu colorimetric assay [18] and expressed as mg gallic acid equivalents (GAE) per g of extract.
Total hydroxycinnamic acid (THC) content was determined by using Arnow's reagent [19] and expressed as mg caffeic acid equivalents (CAE) per g of extract.Total flavonoid (TF) content was determined by using an aluminium chloride colorimetric assay [20] and expressed as mg quercetin equivalents (QE) per gram of extract.Total proanthocyanidin (TPro) content was assessed by the acid-butanol assay [21] and expressed as mg leucocyanidin equivalents (LE) per gram of extract.

Statistical analysis
Experiments were carried out in triplicates.Data presented are mean ± standard error of the mean (SEM).Statistical analyses were performed using Statistical Analysis System (SAS) software (version 9.2).Data were analysed by one-way ANOVA test and means of significant differences were separated using Fisher's least significant difference (LSD) test or Student's t test at α = 0.05.Linear regression and correlation analyses were carried out using Microsoft Office Excel 2007.

Concentration-dependent
increases in αglucosidase inhibitory activity was observed for all the extracts, except D. denticulata (Fig 1).EC 50 values for antiglucosidase activity among the extracts, ranked in ascending order, were D. esculentum < B. orientale < P. vittata (Table 1).The EC 50 value of D. esculentum extract was considerably lower than that of myricetin.The EC 50 values of B. orientale and P. vittata extracts were 24 % and 64 % higher than that of myricetin, respectively.D. denticulata showed αglucosidase activation activity; hence its EC 50 value was not determined.All fern extracts, except D. denticulata, showed dose-dependent cytotoxicity toward K562 cells (Fig 2).The cytotoxic effect of D. denticulata extract decreased with increasing extract concentrations.The EC 50 value for cytotoxic activity of P. vittata was 1.7-fold higher compared with 5-fluorouracil (Table 1).Overall, the five fern extracts investigated contained various levels of phytochemicals (Table 2).Phytochemical analysis revealed that D. denticulata extract had the TP, THC and TF contents, while B. orientale had the highest TPro among the five extracts.There was no significant, positive correlation between the phytochemical parameters and bioactivity with respect to the two parameters analysed in this study.D. denticulata extract was rich in TF (64 % by weight).

DISCUSSION
This study demonstrated for the first time the αglucosidase inhibitory activity of B. orientale, D. esculentum, N. biserrata, and P. vittata.Importantly, despite being a crude extract, the edible fern D. esculentum was a more potent glucosidase inhibitor than myricetin.Our findings are relevant to the current interests in searching for natural antidiabetic agents and managing diabetes by dietary intervention [22,23].The water-soluble and heat-stable nature of the αglucosidase inhibitors in D. esculentum is evident in this study.Such α-glucosidase inhibitors could be readily extracted with water and their activity is likely to be preserved after cooking with heat.
In this study, we assessed the antiglucosidase activity of the extracts by using the yeast αglucosidase.
Yeast α-glucosidase is commercially available in pure form and is often used as a model for evaluating antiglucosidase potential of natural products [15,24,25 To the best of our knowledge, this report is the first account of the cytotoxicity of P. vittata and N. biserrata towards any cancer cell line.Our findings have also added valuable information to the currently limited knowledge on the cytotoxic potential of B. orientale and D. esculentum.Previously, organic and water fractions of methanol extract of B. orientale were found to be non-cytotoxic to K562 cells, although they were cytotoxic to human colonic adenocarcinoma cells (HT-29) and human colonic carcinoma cells (HCT-116) [27].In this study, hot water extract of B. orientale was cytotoxic to K562 cells, although the level of cytotoxicity detected was not as high as that of P. vittata.Ethanolic extract of D. esculentum exhibit no notable cytotoxicity against breast, colon, and liver cancer cell lines [5].Likewise, hydroethanol extract of D. esculentum was not cytotoxic to human cervical carcinoma (HeLa) cell line [28].The present study demonstrated for the first time that D. esculentum was cytotoxic to K562 cells, although its effect is not as potent as that of P. vittata.general, except for D. denticulata, the other four ferns investigated, especially P. vittata, are promising sources of water-soluble and heatstable cytotoxic agents.Future research to isolate and identify cytotoxic constituents from P. vittata will be of great value to therapeutic agent development, especially that for leukaemia treatment.Moreover, the edible ferns B. orientale, D. esculentum, and N. biserrata may be of interest to future research aimed at discovering anticancer drugs of food origin or developing functional food with anticancer potential.
There were no correlations between bioactivity and phytochemical parameters in our study.Phenolic constituents of ferns and other plants are known to exhibit potent antiglucosidase and cytotoxic activities [1,29].Hence, a possible explanation for our observations is that the five fern extracts may contain phenolic constituents that vary considerably in their efficacy or specific activity per unit mass as antiglucosidase or cytotoxic agents.Consequently, their bioactivities are not proportional to and cannot be readily predicted from their total phytochemical contents.Nevertheless, we cannot rule out the possibility that there may be classes of phytochemicals not analysed here which may have contributed to the bioactivities observed in the fern extracts.The nature of the active compounds responsible for the antiglucosidase activity of D. esculentum and the cytotoxicity of P. vittata can only be confirmed when the compounds are isolated from the ferns and structurally characterised.
Our study revealed that D. denticulata extract was rich in flavonoids (64 % by weight).Fern flavonoids are known to have diverse bioactivities [1].

Table 1 :
Antiglucosidase and cytotoxic activities of fern extracts (expressed as EC50), compared with myricetin and 5-fluorouracil Values are mean ± SEM (n = 3).Asterisks (*) denote values that are significantly different (p < 0.05) compared with positive control, as determined using Student's t test.ND = α-glucosidase inhibitory activity was undetectable.
a EC50 was not calculated because maximum activity was below 50 %; b EC50 was not calculated due to lack of increasing trend in activity

Table 2 :
Phytochemical contents of fern extracts cValues are mean ± SEM (n = 3).Values in the same column that are followed by different superscript letters are significantly different (p < 0.05), as determined by using Fisher's LSD test.TP -total phenolics, THC -total hydroxycinnamic acids, TF -total flavonoids, TPro -total proanthocyanidins Thus, such high abundance of flavonoids implies that D. denticulata may possess other bioactivities despite its lack of antiglucosidase and cytotoxic activities.There are no previous reports on the TP contents of D. denticulata.The observed higher TP content of B. orientale than for P. vittata, N. biserrata and D. esculentum are in agreement with the findings of an earlier work[30].