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Abstract—Malaysia is home to a diverse range of ferns, many

of which have been used as traditional medicines in the treatment of various ailments or for general healthcare. The aim of this study was to explore new and natural sources of antioxidant in selected ferns found in Malaysia. Methanolic extracts of fifteen fern species were screened. Total phenolic content (TPC) was measured using the Folin-Ciocalteau method. Antioxidant properties were determined via the DPPH radical scavenging, ferric reducing power (FRP), ferrous ion chelating activity (FIC) and β–carotene bleaching (BCB) assays. Results showed very high total phenolic content, of above 2000 mg GAE/100 g fresh leaves in the methanolic extracts of five fern species. The ferns with strong antioxidant properties were Cyathea latebrosa, Cibotium barometez, Drynaria quercifolia, Blechnum orientale and Dicranopteris linearis. These ferns exhibited strong DPPH radical scavenging activity (ascorbic acid equivalents 2866 – 3111 mg/100 g), ferric ion reducing power (gallic acid equivalents 963 – 1417 mg GAE/100 g) and inhibition of lipid peroxidation. Strong chelating activity was found in Pteris vittata and Pteris venulosa. Positive correlation was observed between TPC and various antioxidant activities.

Index Terms—Antioxidant, DPPH radical scavenging, ferns, polyphenolic compounds

I. INTRODUCTION For a long time, over-production of free radicals has been

claimed to be an exclusively damaging factor that leads to the development of many diseases and various pathophysiological events. Cardiovascular disorders (ischemia-reperfusion, atherosclerosis and hypertension), diabetes mellitus, cancer, anemias, skin inflammation, multiple sclerosis, Alzheimer’s disease, liver diseases, kidney diseases, rheumatoid arthritis and aging are strongly linked to enhanced production of free oxygen radicals [1]. Antioxidants are substances that can delay or prevent the oxidation process of free radicals. They are classified into two groups, namely, primary and secondary antioxidants, depending on their mechanism of action. The former react with lipid peroxy radicals to convert them to stable products; this group includes chain breakers (or free radical inhibitors) and peroxide decomposers. Secondary antioxidants, such as oxygen scavengers, suppress the formation of radicals, hence reduce the rate of chain initiation and protect against

Manuscript received October 18, 2011, revised October 21, 2011. This

work was supported by Monash University Sunway Campus. HowYee Lai is with School of Biosciences, Taylor’s University, 1, Jalan

Taylor’s 47500 Subang Jaya, Selangor, Malaysia (e-mail: HowYee.Lai@ taylors.edu.my).

YauYan Lim is with School of Science, Monash University Sunway Campus, Bandar Sunway, 46150 Petaling Jaya, Selangor, Malaysia (e-mail: [email protected]).

oxidative damage [2]-[4]. The use of synthetic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, tert-butylhydroquinone and propyl gallate has been negatively perceived by consumers due to safety and health effects [5]. Hence, there is an increasing interest in the search of natural antioxidants from plant sources.

It is well known that many botanicals possess natural antioxidants with high antioxidant activity [2]-[6] and investigations on these were initiated based on their uses in traditional folkloric medicines. Ferns belong to a group of non-flowering plants known as Pteridophytes. It is estimated that there are 1136 fern species in Malaysia [7], many of which have been used as traditional medicines in the treatment of various ailments or for general healthcare [8]-[15]. Despite the large number of ferns, knowledge on the antioxidant properties of these botanicals is still scarce. In this study, our main objectives were to investigate the antioxidant properties of fifteen (15) ferns species found in Malaysia. Majority of these ferns have been utilized traditionally to provide cures for many ailments (Table I). Total phenolic content was determined and antioxidant activities were measured based on DPPH radical scavenging, reducing power, β–carotene bleaching assay and metal ion chelating power.

II. MATERIALS AND METHODS

A. Ferns Collection and Extract Preparation Cyathea latebrosa (Wall. ex Hook) Copel, Dicranopteris

linearis (Burm.) and Pteris vittata L. were obtained from Mount Kiara Park, Kuala Lumpur. Cibotium barometz (L.) J. Sm., Drynaria quercifolia (L.) J. Sm., Blechnum orientale L., Adiantum raddianum C. Presl., Diplazium esculentum (Retz.) Sw., Pityrogramma calomelanos (L.) Link, Lygodium circinnatum (Burm.f.) Swartz and Microsorum punctatum (L.) Copel Cv. were collected from Putrajaya Botanical Garden. Nephrolepis biserrata (Sw.) Schott, Pteris venulosa Bl. and Pyrossia numularifolia (Sw.) Ching were collected from private gardens. Acrostichum aureum L. was collected from the Pantai Jeram mangrove swamp in Selangor. The identity of the species were confirmed by plant taxonomist Anthonysamy S., formerly from University Putra Malaysia and currently a consultant with the landscape consulting firm, Aroma Tropic Limited, Kuala Lumpur. Voucher specimens of the ferns were deposited in the herbarium of Monash University Sunway Campus.

Fresh leaves (about 1 g) were pulverized in liquid nitrogen and extracted with 50.0 mL of methanol continuously for an hour at room temperature in a rotary orbital shaker. The extract was filtered under reduced pressure and stored at -20 °C to be used within one week.

Evaluation of Antioxidant Activities of the Methanolic Extracts of Selected Ferns in Malaysia

HowYee Lai and YauYan Lim

International Journal of Environmental Science and Development, Vol. 2, No. 6, December 2011

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TABLE I: FERNS INVESTIGATED AND THEIR ETHNOMEDICINAL USES Fern (family) Ethnomedicinal uses

Acrostichum aureum (Pteridaceae)

Sinus pains, sore throat, for healthy pregnancy, constipation, elephantiasis, febrifuge, chest pain, wounds [10], [11]

Adiantum raddianum (Pteridaceae/ Adiantaceae)

As ornamental plant.

Blechnum orientale (Blechnaceae)

For treatment of boils, blisters, abscesses, sores, wounds, an antihelmintic Tonic for invigoration, impotence [10]

Cibotium barometz (Dicksoniaceae)

Treatment for typhoid, dyspepsia and coughs, tonic for kidney and liver [12], [13]

Cyathea latebrosa (Cyatheaceae) No reports on medical uses.

Dicranopteris linearis (Gleicheniaceae)

Sterility, antihelmintic treatments, antibacterial, asthma, indigestion, hemorrhoids, gastric ulcers, convulsions, appendicitis pain [10]

Diplazium esculentum (Athyriaceae/ Dryopteridaceae

General health tonic and as vegetable [10], [11]

Drynaria quercifolia (Polypodiaceae)

Treatment for typhoid dyspepsia and coughs As an astringent, poultice for swelling. To treat tuberculosis, hectic fever, dyspepsia, phthisis and as an expectorant and antihelmintic. To relieve headache and for bowel inflammation [12], [14].

Lygodium circinnatum (Schizaeaceae)

To neutralize the poison of snake venoms and insects’ sting, wounds [ 9,12]

Microsorum punctatum (Polypodiaceae)

For coughing fits, as an enema [10], [14]

Nephrolepis biserrata (Nephrolepidaceae)

For treatment of blisters, boils, abscesses, sores, wounds, cuts [10]

Pityrogramma calomelanos (Hemionitidaceae)

Used for kidney problem [15]

Pteris venulosa (Pteridaceae) An ornamental plant

Pyrossia nummularifolia (Polypodiaceae)

Cough tonic [8]

Pteris vittata (Pteridaceae)

Whole plant is poisonous; planted on village borders believed to keep enemies away [15]

B. Total Phenolic Content (TPC) TPC in extracts was determined according to the

Folin-Ciocalteu procedure [16]. The extract (0.3 mL, in triplicate) was mixed with 1.5 mL of 10% Folin-Ciocalteau’s reagent and 1.2 mL of 7.5% (w/v) sodium carbonate. The mixture was kept in the dark for 30 min before measuring the absorbance at 765 nm. A calibration curve was constructed using gallic acid and results were expressed as mg gallic acid equivalent (GAE)/100g leaves.

C. Antioxidant Assays DPPH radical scavenging activity. The DPPH radical

scavenging assay employed is as previously described [17]. Various dilutions of the extract (1.0 mL, in triplicate) were added to 2.0 mL of DPPH (5.9 mg/100 mL methanol). The mixture was left in the dark for 30 min before reading the absorbance at 517 nm with methanol as blank. The control consisted of methanol in place of extract. Radical scavenging activity was expressed as a percentage and calculated using the formula: %Scavenging = (Acontrol - Asample)/Acontrol x 100. Result was presented as IC50, the concentration of extract

required to scavenge 50% of the DPPH radical and also in terms of ascorbic acid equivalent antioxidant capacities (AEAC) which was calculated as follows: AEAC (mg AA/100g) = IC50 (ascorbic acid)/IC50 sample x 105. IC50 (ascorbic acid) was determined to be 0.00387 mg/mL.

Ferric ion reducing power (FRP). The Ferric Reducing Power (FRP) assay was carried out according to the procedure described previously [4]. Various dilutions of the extract (1.0 mL , in triplicate) were added to 2.5 mL of 0.2 M phosphate buffer pH 6 and 2.5 mL of potassium ferricyanide (1% w/v). The mixture was incubated for 20 min at 50°C, after which 2.5 mL of 10% trichloroacetic acid was added. An aliquot of 2.5 mL of each mixture was diluted twice with deionised water, before adding 0.5 mL of 0.1% (w/v) FeCl3. Absorbance was measured at 700 nm after 30 min. A calibration curve was constructed using gallic acid. Results were expressed as mg gallic acid equivalent (GAE)/100 g leaves.

Beta-carotene bleaching (BCB) assay. The assay was carried out as described previously [4]. A stock solution of β-carotene/linoleic acid was initially prepared by dissolving 5 mg of β-carotene in 50 mL of chloroform. An aliquot of the β-carotene solution (3 mL) was added to 40 mg of linoleic acid and 400 mg of Tween 40. The chloroform was evaporated off using nitrogen gas. Aerated distilled water (100 mL) was added to the mixture. An initial absorbance at 470 nm and 700 nm was immediately recorded. Aliquots of β-carotene/linoleic acid emulsion (3 mL) were mixed with 10 µL, 50 µL and 100 µL of extract. The test and control (containing water in place of sample) tubes were capped and incubated at 50°C. The absorbance of the emulsion at 470 nm and 700 nm was determined after 60 min. All determinations were performed in triplicate. The antioxidant activity was calculated using the formula: Degradation rate (DR) of β-carotene = Ln(Ainitial/Asample)/60. Antioxidant activity (%AOA) = [(DRcontrol – DRsample)/DRcontrol] x 100.

Ferrous ion chelating (FIC) activity. The procedure has been described previously [18]. Various dilutions of the extract solution (1.0 ml, in triplicate) were added to 1.0 ml of 0.1 mM FeSO4 and 1.0 ml of 0.25 mM ferrozine. The tubes were shaken well and left to stand for 10 min. The absorbance was measured at 562 nm, against a blank containing water in place of ferrozine. The control consisted of water in place of the extract. The ability of a sample to chelate ferrous ion was calculated as follows: chelating effect (%) = (Acontrol- Asample)/Acontrol x 100.

III. RESULTS AND DISCUSSION Phenolics are the most widespread secondary metabolites

in the plant kingdom and have received much attention as potential natural antioxidants in terms of their abilities to act as both efficient radical scavengers and metal chelators [2]-[6]. In this study, fifteen (15) ferns were investigated for the total phenolic content and their antioxidant properties.

A. Total Phenolic Content (TPC) Wide variation in TPC was observed amongst the ferns as

shown in Fig. 1. Based on the values of TPC, the ferns were


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divided into four categories: (a) ferns with very high TPC i.e. > 2000 mg GAE/100 g leaves: Cyathea latebrosa, Cibotium barometz, Drynaria quercifolia, Dicranopteris linearis and Blechnum orientale, (b) ferns with high TPC i.e. 1000 – 1999 mg GAE/100 g leaves: Adiantum raddianum and Pteris vittata, (c) ferns with moderate TPC i.e. 500 – 999 mg

GAE/100 g leaves: Acrostichum aureum, Nephrolepis biserrata, Diplazium esculentum, Pityrogramma calomelanos, Lygodium circinnatum and Pyrossia nummularifolia and (d) ferns with low TPC i.e. < 500 mg GAE/100 g leaves: Pteris venulosa and Microsorum punctatum.

Fig. 1. Total phenolic content (TPC) of methanolic leave extracts from fifteen fern species. Very high TPC; High TPC; moderate TPC; low TPC. Data are mean ± S.D of 3 samplings collected randomly in 3 batches from the same source (n=9). Triplicate measurements were conducted in each assay.

Phenolic compounds are generated by plants in response to environmental stress. It has been reported that light stimulates the synthesis of flavonoids, especially anthocyanins and flavones via phenylalanine ammonia lyase (PAL) [19] and phenolics are thought to provide a means of protection against UV-B damage and subsequent cell death by protecting DNA from dimerization and breakage [20]. Therefore plants in high-mountain areas which are exposed to a number of stress factors such as low air temperature, decreased partial O pressure, increased UV radiation and unfavorable water regime have generally increased accumulation of antioxidants such as flavonoids [21]. This partially explains the exceptionally high TPC in the ferns with very high TPC (C. latebrosa, C. barometz, D. quercifolia, B. orientale and D. linearis) and high TPC (A. raddianum and P. vittata), as they can grow well in habitats of exposed sunlight and at slopes or mountain regions with altitudes up to 1500 – 1700 m.. Salinity is the primary environmental stress factor for a mangrove plant such as A. aureum and hence high TPC was expected for this fern. However, A. aureum showed moderate TPC (945 mg GAE) which could be due to the shade-tolerant ability of this fern. It has been reported that shades help to reduce the rate of evaporation hence relieving the salt stress encountered by A. aureum [22]. For an epiphyte such as P. nummularifolia, climbing on tree trunk provides an environment with lower extremes in temperature and sun illumination.

B. DPPH Scavenging, Ferric Reducing Power (FRP) and Beta-Carotene Bleaching (BCB) In the evaluation of the primary antioxidant activity, DPPH

scavenging activity, ferric reducing power (FRP) and BCB methods have been widely used [3]-[6], [17]. In DPPH scavenging assay, the antioxidant activity was measured by the decrease in absorbance as the DPPH radical received an electron or hydrogen radical from an antioxidant compound

to become a stable diamagnetic molecule [4]. The antioxidant activity was expressed as IC50 (concentration of the antioxidant required to scavenge 50% of the initial DPPH radicals) and AEAC (Ascorbic acid equivalent antioxidant capacity). The lower the IC50 and the higher the AEAC value, the greater is the antioxidant activity. The FRP method measured the ability of an antioxidant to donate electron to Fe (III) resulting in the reducing of Fe3+/ferricyanide complex to Fe2+ complex, which could be monitored at 700 nm. Results were expressed as mg gallic acid equivalent (GAE)/100 g leaves. The higher the FRP value, the greater is the reducing power of the tested compound, thus the greater the antioxidant activity.

Generally, the order of the DPPH scavenging and FRP activities displayed by the ferns were similar to that of TPC i.e. ferns with very high TPC (AEAC 2821 – 3200 mg AA/100g and FRP 963 – 1466 mg GAE/100g) > ferns with high TPC (AEAC 1438 – 1645 mg AA/100g and FRP 578 – 842 mg GAE/100g) > ferns with moderate TPC (AEAC 225 - 740 mg AA/100g and FRP 201 - 578 mg GAE/100g) > ferns with low TPC (AEAC 157 - 197 mg AA/100g and FRP 121-176 mg GAE/100g) (Table II).

BCB method measured the ability of an antioxidant to inhibit lipid peroxidation. In the BCB method, a model system made of β-carotene and linoleic acid undergoes a rapid discoloration in the absence of an antioxidant. The free linoleic acid radical formed upon the abstraction of a hydrogen atom from one of its methylene groups attacked the β-carotene molecules, which lost the double bonds and therefore, its characteristic orange color. The rate of bleaching of the β-carotene solution was measured by the difference between the initial reading in spectral absorbance at 470 nm at time 0 min and after 60 min [4]. The antioxidant activity was expressed as percent inhibition relative to the control. It can be observed from Fig. 2 that the BCB

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antioxidant activity increased with increasing concentration of the extract used. D. linearis exhibited the highest BCB antioxidant activity amongst the ferns tested, with 61% at 0.1 mg/mL and extended to 99% at 0.7 mg/mL. The other ferns of high and very high TPC namely A. raddianum, C. latebrosa, D. quercifolia, B. orientale and C. barometz also

showed strong activity of 64-88% at 0.7 mg/mL. These results implied that the potential antioxidant capabilities in C. latebrosa, C. barometz, D. quercifolia, B. orientale and D. linearis were attributed to the phenolic compounds in these fern species.

TABLE II: DPPH SCAVENGING ACTIVITY (EXPRESSED IN IC50 AND AEAC) AND FERRIC REDUCING POWER (FRP) OF THE METHANOLIC EXTRACTS OF FIFTEEN FERN SPECIES

DPPH scavenging activity FRP Fern Parts taken IC50 (mg/ml) AEAC (mg AA/100 g) (mg GAE/100g) Ferns with very high TPC C. latebrosa leaves 0.13 ± 0.03 3111 ± 722 1417 ± 268 C. barometz leaves 0.12 ± 0.01 3200 ± 336 1466 ± 81 D. quercifolia leaves 0.14 ± 0.03 2835 ± 448 1091 ± 107 B. orientale leaves 0.14 ± 0.03 2821 ± 705 1089 ±157 D. linearis leaves 0.14 ± 0.02 2866 ± 433 963 ± 63 Ferns with high TPC A. raddianum leaves 0.27 ± 0.02 1438 ± 110 842 ± 46 P. vittata leaves 0.29 ± 0.03 1645 ± 499 578 ± 97 Ferns with moderate TPC A. aureum leaves 0.54 ± 0.04 724 ± 59 524 ± 20 D. esculentum leaves 0.57 ± 0.12 700 ± 132 578 ± 49 P. calomelanos leaves 1.20 ± 0.18 329 ± 58 364 ± 27 P. nummularifolia leaves 1.47 ± 0.08 264 ± 15 201 ± 13 N. biserrata leaves 0.53 ± 0.05 740 ± 71 422 ± 46 L. circinnatum leaves 1.73 ± 0.12 225 ± 16 220 ± 9 Ferns with low TPC: P. venulosa leaves 1.99 ± 0.28 197 ± 29 176 ± 12 M. punctatum leaves 2.51 ± 0.42 157 ± 25 121 ± 26

Data are mean ± S.D of 3 samplings collected randomly in 2 batches from the same source (n=6). Triplicate measurements were conducted in each assay. TPC, total phenolic content; AEAC, ascorbic acid equivalent antioxidant capacity; FRP, ferric ion reducing power.

Fig. 2. β-carotene bleaching (BCB) activities of six fern species with high to very high TPC (left) and of four fern species with low to moderate TPC (right).

C. Ferrous Ion Chelating (FIC) Fe2+ has been known to accelerate formation of

hydroxyl radicals via the Fenton reaction, leading to occurrence of many diseases [1], [4]. It is reported that chelating agents that form σ-bonds with a metal ion, are effective secondary antioxidants since they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion [23]. In the FIC assay, ferrozine acts as a chelating agent and forms purple complex ion with Fe2+ giving maximum absorbance at 562 nm. Fig. 3 shows the chelating activity of the ferns.

Generally, the chelating activity of all ferns increased with

increasing concentrations of the extract used. Amongst the ferns with very high and high TPC, only P. vittata and A. raddianum showed relatively high chelating activity of 61.3% and 78.3% respectively at concentration 6.7 mg/mL. However, other ferns in this category showed relatively weak chelating power (16% to 32% at 6.7 mg/mL). On the other hand, ferns with moderate TPC exhibited relatively high chelating activity of 34 -67% at the same concentration. It is interesting to note that P. venulosa with low TPC, gave minimal chelating activity (<10%) at lower concentrations but increased sharply to a high activity of 73.5% at 6.7 mg/mL.

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Fig. 3. Chelating activity (%) of ferns with very high and high TPC (upper diagram), moderate TPC (middle diagram) and low TPC (lower diagram)

D. Correlation between TPC and Antioxidant Activities Analysis on the correlations between TPC and the

antioxidant properties measured showed good correlations between TPC with AEAC (R2=0.953) and FRP (R2=0.943). These data suggest that phenolic compounds are powerful scavenger of free radicals as well as reducing agents. Similar trend was reported in earlier studies [5], [17], [23]. Analysis on the TPC and BCB data revealed moderate correlation (R2=0.663) between these parameters which is in agreement

with an earlier report [24]. Poor correlation (R2=0.371) found between TPC and FIC indicates that some phenolic compounds in the plant extract are weak ion chelators. Based on data from DPPH, FRP, BCB and chelating activity assays, it can be deduced that high primary antioxidant activity (measured by DPPH, FRP and BCB assays) may not necessarily correlate with its secondary antioxidant activity (measured by FIC assay), indicating different mechanisms of action in the phenolic compounds of ferns. Amongst the ferns, five ferns possessing very high phenolic compounds namely C. latebrosa, C. barometz, D. quercifolia, B. orientale and D. linearis are strong potent primary antioxidants but weak secondary oxidants. P. vittata and A. raddianum showed both high primary and secondary antioxidant activity. This indicates that the phenolic compounds in these ferns are potent radical scavengers, radical chain breakers and peroxide decomposers. The high chelating activity also indicates that these ferns have potential in reducing the chance of chain initiation by chelating iron (II) and hence preventing oxidative damage. In contrast, P. venulosa which showed weak primary antioxidant activity from its high IC50 and low FRP data, surprisingly exhibited strong chelating activity (> 70%). This indicates that this fern may be potent secondary antioxidants, though weak primary antioxidant. Similar trends were observed by Lim et al (2007) in several tropical fruits [3].

The difference in the correlations between TPC and antioxidant assays indicates the diversity of the group of phenolic compounds in the ferns and their different responses to different methods for the determination of the antioxidant activity. A reason for this difference is due to the fact that Folin-Ciocalteau method determined the sum of phenolic compounds, whereas individual phenolic compounds have very different responses on the Folin-Ciocalteau reagent and made different contributions to the antioxidant activity [25]. This is in agreement with the findings by Goupy et al. (1999) [24] who tested the antioxidant activity of individual phenolic compounds and found that flavan-3-ols possessed the highest radical scavenging activity while the mechanism of antioxidant activity depended on specific structures e.g. the number of hydroxyl groups and the presence of the -CH=CH-COOH group. For a deeper insight on the effects of the extracts on the methods for the determination of the antioxidant activity, it would be necessary to characterize the individual phenolic compounds.

IV. CONCLUSION Methanol extracts of C. latebrosa, C. barometez, D.

quercifolia, B. orientale and D. linearis showed very high total phenolic content and are potent primary antioxidants as shown by their high radical scavenging capacity, reducing activity and BCB antioxidant activity. In contrast, extracts of P. vittata and P. venulosa are potent secondary antioxidants due to their strong chelating power. Amongst these potent ferns, C. barometz, D. quercifolia, B. orientale and D. linearis are commonly used in traditional healing. Further investigative work has been carried out on B. orientale and is reported elsewhere [26].

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ACKNOWLEDGMENT The authors would like to thank the administrators of

Putrajaya Botanical Garden, Kuala Lumpur for the kind contribution of ferns. This project was conducted at Monash University Sunway campus. The financial grant from the university is acknowledged.

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