Antioxidant potential of Amorphophallus paeoniifolius in relation to their phenolic content

Abstract

The present study evaluates the antioxidative and radical scavenging potential of the tuber extract of Amorphophallus paeoniifolius (Dennst.) Nicolson, (Araceae). The ethanol extract of A. paeoniifolius (APE) was studied for the inhibition of lipid peroxidation estimated in terms of thiobarbituric acid reactive substances (TBARS) and the levels were reduced by 4.3% to 67.2% in a dose-dependent manner. Further, APE was analyzed for scavenging capacities based on 1,1-diphenyl-2-picrylhydrazyl-2-radical (DPPH) assay and percentage inhibition activity based on 2,2-azinobis-(-3-ethyl) benzo-thiozoline-6-sulfonate (ABTS⁺) and H₂O₂. The A. paeoniifolius extract showed a maximum of 68.6% of DPPH scavenging activity and the maximum inhibition of 74% and 67.2% in the case of ABTS and H₂O₂, respectively. The antioxidant efficiency and inhibition of oxidation of the extract was found to be dose-dependent at the tested concentrations of 1-50 µg/mL. High-performance thin layer liquid chromatography (HPTLC) profile of the extract suggests the presence of polyphenols such as gallic acid, resveratrol, quercetin and two unidentified compounds. The results suggest that the ethanol extract of A. paeoniifolius has a potent antioxidant activity in vitro and can be utilized as an effective and safe source of antioxidants.

Keywords::

• Amorphophallus paeoniifolius
• antioxidant
• polyphenols
• free radicals
• medicinal plants

Introduction

India is considered to be a rich emporium of medicinal plants, mainly used in preventive and curative medicine. Over 50% of all modern clinical drugs are of natural product origin and they play an important role in drug development programs of the pharmaceutical industry (Baker et al., 1995). In recent years, the popularity of complementary medicine has increased. Dietary measures and traditional plant therapies as prescribed by Ayurvedic and other indigenous systems of medicine are used commonly in India (Warier, 1995). The World Health Organization (WHO, 2002) has also recommended the evaluation of the plants effective in conditions where safe modern drugs are lacking. An intensive search for novel types of antioxidants has been carried out from numerous plant materials (Scartezzini & Speroni, 2000; Ng et al., 2000).

A large number of synthetic antioxidants, such as Butylated hydroxytoluene (BHT) and Butylated hydroxyanisole (BHA) have been reported as chemopreventive agents for cancer, but there have been reports of adverse effects (Kahl & Kappus, 1993; Botterweck et al., 2000). Hence, the natural antioxidants available in our food source have been suggested as safe for therapy (Athar, 2002). A number of antioxidants have been derived from plants and have been widely studied (Balunas & Kinghorn, 2005; Dehghan et al., 2007; Puvanendran et al., 2008; Rosidah et al., 2008). Numerous studies have also indicated that many plant products, including flavonoids, anthraquinones, tannins, proanthocyanidins, and other phenolics, as well as substances derived from fruit, vegetables, and various plant or herbal extracts, have radical-scavenging activity (Borek, 1997). Therefore, these substances could be proposed as health-beneficial products.

Amorphophallus paeoniifolius (Dennst.) Nicolson (Araceae) is a commonly available tuber in South India, widely used in folk medicine for treatment of acute rheumatism, tumors, lung swelling, asthma, vomiting, and abdominal pain (Bogner, 1995). So far, no attempts have been made to evaluate the chemical composition and medicinal properties of A. paeoniifolius. Hence, the present study was performed to investigate the antioxidant potential of ethanol extract of A. paeoniifolius using different in vitro models, and its polyphenolic composition was also determined.

Materials and methods

Chemicals

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2-azinobis-(-3-ethyl) benzo-thiozoline-6-sulphonate (ABTS), butylated hydroxy toluene (BHT) and nitroblue tetrazolium (NBT) were obtained from Sigma, St. Louis, MO. All other chemicals and solvents used in the study were obtained from SD-Fine, Mumbai, India.

Plant material

Amorphophallus tuber was collected from a local market in Coimbatore, Tamil Nadu during July, 2004. The plant specimen was authenticated by R. Gopal (Botanical Survey of India, Southern Circle, Coimbatore, Tamil Nadu, India). A voucher specimen (SP No. 1) was deposited at the herbarium unit of the Biotechnology Department, Bharathiar University, Coimbatore (BU/BT/SPE-24).

Tuber extract preparation

Amorphophallus tuber (250 g) was dried under shade and the yield obtained was 32.5% w/w. The dried material was ground to a fine powder (particle size ∼0.25 mm) using a laboratory mill and first defatted with 750 mL petroleum ether (60°–80°C) for 2-3 h in a Soxhlet apparatus, the resulting extract was dried and extracted with 1 L ethanol (80%) in a Soxhlet apparatus for 5 h. The extract was filtered, subjected to concentration at a reduced pressure using a rotary evaporator and subjected to lyophilization to obtain a crude ethanol extract (Wilson et al., 2005). The percentage yield was found to be 6.2%. The extracted fraction was used for analyzing the antioxidant properties.

Inhibition of free radical generation by oxidation

The oxidation inhibiting effect of the extract to prevent free radical formation was tested by performing the lipid peroxidation assay in vitro using rat brain homogenate.

Preparation of rat brain homogenate

Adult male Wistar rats weighing about 200-250 g were used. The animals were fed ad libitum with normal laboratory pellet diet (Hindustan Lever, Mumbai, India) and water. Animals were maintained under a constant 12 h light and dark cycle and at an environmental temperature of 21–23°C. The animals used in the present study were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. The rats were deprived of food 12 h prior to sacrifice. The animals were euthanized and sacrificed by cervical decapitation, dissected and the whole brain except cerebellum was removed quickly. It was further processed to get 10% homogenate in 0.15 M KCl using a Teflon homogenizer (Auddy et al., 2003). The homogenate was filtered to get a clear solution and used as a source of polyunsaturated fatty acids for determining the extent of lipid peroxidation.

In vitro lipid peroxidation determination

The extent of lipid peroxidation in rat homogenate was measured in vitro in terms of formation of thiobarbituric acid reactive substances (TBARS). Different concentrations of the extract were made up with ethanol and expressed in terms of dry weight (µg/mL). The samples were individually added to the brain homogenate (0.5 mL). This mixture was incubated with 0.15 M KCl (100 μL). Lipid peroxidation was initiated by adding 100 μL of 15 mM FeSO₄ solution. The reaction mixture was incubated at 37°C for 30 min. An equal volume of TBA: TCA (1:1) reagent was added to the above solution followed by the addition of 1 mL BHT. This final mixture was heated over a water bath for 20 min at 80°C and cooled, then centrifuged and the absorbance was read at 523 nm using a spectrophotometer (Ohkawa et al., 1979). The percentage inhibition of lipid peroxidation was calculated by comparing the absorbance of the test with those of controls not treated with the extract as per the formula:

Antioxidant potential

Antioxidant potential of the extract of A. paeoniifolius tuber was analyzed based on its capacity to scavenge DPPH, ABTS and H₂O₂. The extract was tested at different concentrations from1-50 µg/mL. In our preliminary studies, we examined antioxidant properties of the extracts obtained using different solvents and found that ethanol extract showed higher antioxidant efficiency along with the high conten)t of phenolic compounds. Hence, we carried out the study using several in vitro model systems to study the antioxidant activity of ethanol extract of this plant.

Antioxidant assay with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical

DPPH is a free radical which when dissolved in ethanol has a blue-violet color. When it reacts with the reducing agent, the solution loses color which depends upon the number of electrons taken up. Hence, the loss of color indicates radical scavenging activity of test material. DPPH was measured by the method of Cervato et al. (2000). Three ml of 60 μM DPPH in ethanol was added to 1 mL of the extract and then incubated at room temperature for 15 min. Absorbance was read at 517 nm using a Genesys-5-spectrophotometer (Spectronic Instruments, Rochester, NY). The antioxidant activity was calculated as inhibition (%) of DPPH radical formation:

ABTS⁺ cation radical and antioxidant activity

The total antioxidant activity of the extract was measured by the ABTS radical cation-decolorization assay (Pellegrini et al., 2003). ABTS was prepared by reacting 5 mL of 7 mM ABTS and 88 μL of 140 mM potassium persulfate and the mixture was allowed to stand in the dark at room temperature for 12-16 h before use. ABTS (1 mL) was added to glass test tubes containing 50 μL of the extract and mixed by vortex mixer for 30 s. Absorbance was measured at 734 nm after 2 min. The percentage inhibition was calculated by:

Determination of H₂O₂ scavenging activity

The ability of extracts to scavenge H₂O₂ was determined according to the method of Ruch et al. (1989). Extracts at different concentrations in ethanol were added to H₂O₂ solution (0.6 mL, 40 mM). Absorbance of H₂O₂ at 230 nm was determined 10 min later against a blank solution containing the phosphate buffer without H₂O₂. The percentage of H₂O₂ scavenging of both the extracts and standard compounds were calculated:

Chemical characterization of A. paeoniifolius using HPTLC

Total phenolic assay

Total phenolics were determined according to the method of Rajeshwar et al. (2005). One mL of the extract was taken into test tubes and mixed with 1 mL 95% ethanol, 5 mL water and then 0.5 mL 1 N Folin-Ciocalteu reagent was added. After 5 min, 1 mL of 5% Na₂CO₃ was added and the reaction mixture was allowed to stand for 60 min before the absorbance at 765 nm was measured. A standard curve was established for each assay using 50 to 500 μg of gallic acid in 95% ethanol and expressed as gallic acid equivalent (1mg gallic acid equivalent per g leaf extract) (Rajeshwar et al., 2005; Tabernero et al., 2006).

Estimation of polyphenolics using HPTLC

The extract was dissolved in ethanol at a concentration of 1 mg/mL and 10 µL of the aliquot was spotted over HPTLC silica gel plate with a 100 µM Silica coating (Merck KGaA-silica gel 60F 254, Darmstadt, Germany) at 12 mm from the lower edge using an automatic TLC Sampler III apparatus (winCATS Planar Chromatography Manager) as bands of 10 mm length. After 10 min, the chromatogram was developed with toluene: ethyl acetate: glacial acetic acid (12.5: 7.5: 0.5 v/v) as mobile phase. The chromatogram was run at 48°C until the mobile phase traveled approximately 16 cm in distance. Then the plates were air dried for 10 min and scanned at 260 nm. Gallic acid, resveratrol, and quercetin were used as standards and run along with the sample. The compounds were quantified using a calibration curve obtained by plotting the concentration of standard against the peak area on the scanned chromatogram (Vanhaelen-Fastre et al., 2000).

Results

Antioxidant activity of A. paeoniifolius tuber extract

The ethanol extract of A. paeoniifolius tuber shows moderate inhibitory activity against lipid peroxidation which was substantiated by the low levels of TBARS value. The extract showed a percentage inhibition ranging between 4.3 to 67.2% in a dose-dependent manner (Table 1).

Table 1.  Effect of alcoholic extract of Amorphophallus paeoniifolius on inhibition of lipid peroxidation and H₂O₂ scavenging activity. (Values are mean of three replicates.)

Concentration of extract (μg/ mL)	Inhibition (%)
	Lipid peroxidation	H2O2 scavenging activity
50	57.5	67.2
40	49.6	61.7
30	42.5	53.8
20	35.4	46.5
10	24.5	30.2
5	12.6	16.3
1	7.2	4.3

Radical scavenging activity of A. paeoniifolius tuber extract

DPPH scavenging activity

The scavenging activity on DPPH was found to be dose-dependent (Figure 1). The extract at the concentration of 50 µg/mL had higher inhibitory activity of 68.6%. The IC₅₀ value was observed to be 30 µg/mL. The results reveal that extracts contain powerful inhibitor compounds, which may act as primary antioxidants that react with free radicals.

Figure 1.  Scavenging effects of A. paeoniifolius extract (APE) on DPPH radical. Butylated hydroxyl toluene (BHT) was used as a positive control. Values represent the mean ± SD (n = 3).

ABTS⁺ scavenging activity

The percentage inhibition of ABTS⁺ was also found to be dose-dependent (Figure 2) suggesting the sensitivity of the assay towards ethanol-soluble antioxidants. The IC₅₀ value of the extract was found to be 35 µg/mL. The results were compared with the standard BHT, as a reference compound.

Figure 2.  Scavenging effects of A. paeoniifolius extract (APE) on ABTS radical. Butylated hydroxyl toluene (BHT) was used as a positive control. Values represent the mean ± SD (n = 3).

H₂O₂ scavenging activity

Tuber extract at 40 µg/mL exhibited 50% scavenging activity. The same patterns of results were observed as earlier, which may suggest the possible high level of lipophilic phenolic antioxidants in the extract (Table 1).

Chemical characterization of A. paeoniifolius tuber extract

Total phenolic composition

The total phenolic content in A. paeoniifolius at different concentrations was analyzed by the Folin-Ciocalteu method (Figure 3). The phenolic content of the extract was found to be 44.58 mg GAE/g powder. The total phenolic content was correlated with the inhibition of lipid peroxidation and was represented in Figure 4. A highly significant positive correlation was observed between the phenolic content and the antioxidant activity measured by in vitro lipid peroxidation.

Figure 3.  Presence of total phenolic content in A. paeoniifolius (APE). The phenolic content of the extract was found to be 44.58 mg GAE/g powder. Values represent the mean ± SD (n = 3).

Figure 4.  Relationship between antioxidant activity and total phenolic content in A. paeoniifolius.

HPTLC analysis

The preliminary phytochemical testing indicated the presence of high amounts of phenolics along with flavonoids and alkaloids (unpublished data). The HPTLC analysis showed five major peaks having Rf values 0.06, 0.29, 0.41, 0.47, and 0.95 (Figure 5). The Rf values of 0.06, 0.41, and 0.47 peaks are consistent with those of gallic acid, resveratrol, and quercetin standards respectively. The content of gallic acid was found to be 6.41% w/w resveratrol was 1.8% w/w and quercetin was 0.96% w/w. The other peaks are remained to be identified.

Figure 5.  The HPTLC chromatogram of Amorphophallus paeoniifolius leaf extract at 260 nm. The chromatogram shows five major peaks when scanned at 260 nm using toluene: ethyl acetate: glacial acetic acid (12.5: 7.5: 0.5 v/v) as mobile phase. The three peaks (1, 3 and 4) of Rf values are gallic acid (0.06), resveratrol (0.41) and quercetin (0.47), respectively, and two others (2 and 5) remain to be identified.

Discussion

Plant substances continue to serve as a viable source of drugs for the world population and several plant-based drugs are in extensive clinical use (Roja & Rao, 2000). For the past few decades, a number of plants have been widely used for the treatment of various diseases due to their antioxidant properties (Saeed et al., 2005; Kahkonen et al., 1999). The plants described contain antioxidant principles that can explain and justify their use in traditional medicine in the past as well as the present. For the preparation of the herbal mixture of Indian traditional medicine, the antioxidant activity arising from their content of plants due to their antioxidant principles is very important (Scartezzini & Speroni, 2000). A. paeoniifolius has already been listed as a medicinal plant and widely used in Ayurveda therapy (Pal, 2000). In order to identify the plants with antioxidant activity in Indian traditional medicine, this study was undertaken to examine the antioxidant properties of A. paeoniifolius tuber, which has been used traditionally for many years.

In this research, the TBARS assay of the ethanol extract of A. paeoniifolius tuber measured the ability of the antioxidant to prevent the oxidative degradation of lipids. The observed inhibition of the oxidation suggests the presence of a biologically functional compound preventing the oxidative degradation of membrane lipids.

It has been suggested that the free radical responsible for toxicity is the hydroxyl radical, formed via the metal catalyzed Haber-Weiss reaction or Fenton reaction (Lubec et al., 1996; Rinne et al., 2003). In this process, the ferric iron is reduced by superoxide, with subsequent oxidation of ferrous iron by H₂O₂ forming a hydroxyl radical thereby initiating the series of oxidative reactions. The antioxidative property of this plant may be attributed to a number of reasons including the scavenging of hydroxyl or superoxide radical, by changing the ratio of Fe³⁺/Fe²⁺, reducing the rate of conversion of ferrous to ferric or by chelation of iron (Braughler et al., 1986).

The antioxidant potential of the extract was measured by its scavenging activity of DPPH, ABTS⁺, and H₂O₂. The DPPH radical scavenging activity suggested that the physico-chemical nature of the individual phenolics present in the extract may be more important in contributing to the antioxidant activity than the total phenolic content measured by the Folin-Ciocalteu assay. The scavenging of DPPH is representative of the capacity of test compounds to scavenge free radicals independently from any enzymatic activity. The phenolic compounds present in the extract such as gallic acid, resveratrol and quercetin, possibly contributes to the scavenging activity on DPPH radicals. BHT, a reference drug, presented the highest DPPH radical-scavenging activity at all concentrations with IC₅₀ value of 17 µg (Figure 1).

In the ABTS⁺ radical cation-decolorization assay, the extract showed potent scavenging ability that suggests the presence of phenolic antioxidants in the extract. However, the activity was found to be less than the reference drug. The scavenging effect on DPPH radicals and ABTS represent direct radical-scavenging activity. Moreover, the scavenging effect of the extract was directly correlated with their suppressive effect on lipid peroxidation.

The antioxidant activity of plant extracts varies with assay methods (Sun & Ho, 2005). Therefore, a single assay may be inadequate (Yen et al., 2005). For this reason we cross checked antioxidant activities of A. paeoniifolius tuber with different antioxidant activity assays based on different mechanisms. In our preliminary studies we examined the antioxidant property of the extracts obtained using different solvents and found that ethanol extract showed higher antioxidant efficiency along with the high content of phenolic compounds. This finding coincides with a previous study that reported the extraction using ethanol or methanol exhibited the highest total phenolic content, followed by water and ethyl acetate (Jayaprakasha et al., 2008). Based on the difference in polarity of the extraction solvents, the solubility of phenolic compounds and the rate of mass transfer could be different (Bi et al., 2009). As flavonoids and phenolic acids are more soluble in ethanol, it is reasonable to obtain a higher extraction yield when ethanol is used (Markham, 1982).

The antioxidative effect of this extract is mainly due to the presence of phenolic components, such as flavonoids, phenolic acids, and phenolic diterpenes. The antioxidant activity of phenolic compounds is possibly due to their redox properties, which plays an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. Many of these phytochemicals possess significant antioxidant capacity that may be associated with lower incidence and lower mortality rates of cancers and other oxidative stress-related disorders in human populations. Numerous phenolic phytochemicals have been reported for antioxidative, anticarcinogenic, antimicrobial, antiallergic, antimutagenic, antidiabetic, and anti-inflammatory activities (Ho, 1992). Among the numerous polyphenols, gallic acid, resveratrol, and quercetin are widely distributed in the plant kingdom and reported to possess antioxidant, antidiabetic, and anticancer properties (Bravo, 1998). The content of gallic acid was found to be 6.41% w/w, resveratrol was 1.8% w/w, and quercetin was 0.96% w/w. The phenolic antioxidants may contribute directly to antioxidative property as free radical terminators as well as metal chelators (Rice-Evans et al., 1997). The total phenolic content of 1 g of the extract was found to be 44.58 mg GAE. The phenolic composition present in the extract may be a possible reason for the antioxidant potential.

Based on our observations, it may be concluded that the A. paeoniifolius tuber possesses significant antioxidant potential and these effects were attributed to their phenolic constituents. Hence, ethanol extracts may be used as a potential dietary antioxidant to retard aging and prevent diseases caused by ROS or ameliorating oxidative damage.

Acknowledgements

We thank Dr. Jayabal Panneer Selvam, Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, for scientific contributions and helpful discussions.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.