Antioxidant Properties and Heavy Metal Content of Lotus Plant

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1 Food Sci. Technol. Res., 18 (3), , 2012 Antioxidant Properties and Heavy Metal Content of Lotus Plant (Nelumbo nucifera Gaertn) Grown in Ex-tin Mining Pond near Kampar, Malaysia Ee Shan Leong, Sally Tan and Ying Ping Chang * Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, Kampar, Perak, Malaysia Received January 11, 2012; Accepted February 27, 2012 Lotus plant (Nelumbo nucifera Gaertn) is well known for its therapeutic effects despite its ability to accumulate heavy metals. This study evaluates antioxidant properties and heavy metal content of seed, stem and rhizome of lotus that grown in ex-tin mining pond. Total phenolic content, DPPH-radical scavenging activity and tyrosinase inhibition activity were determined on methanol extracts. Contents of ash, lead and chromium were determined on dried, ground samples. The highest concentration of phenolic content, ash, lead and chromium is in the seeds. Tyrosinase inhibition activity is highest in the rhizome and lowest in the stem. Correlation of DPPH-radical scavenging activity is good with tyrosinase inhibition activity (P < 0.01). The concentration of lead found exceeded the permissible limits of Codex Alimentarius. Therefore, assessment of metal contamination should be a mandatory priority to utilization of abandoned ex-tin mining area for edible plant cultivation. Keywords: antioxidant properties, heavy metal content, ex-tin mining pond, lotus plant, Nelumbo nucifera Introduction Lotus (Nelumbo nucifera Gaertn) which is also known as sacred lotus or Chinese arrowroot, is a valued aquatic plant grown and consumed throughout Asia. Almost every part of the lotus plant from stamens, pollens, flowers, stems, rhizomes to leaves can be consumed as food, or for various therapeutic purposes in India and China (La Cour et al., 1995; Sridhar and Bhat, 2007; Mukherjee et al., 2009). For example, lotus seeds are good source of protein and essential minerals (Ibrahim and El-Eraqy, 1996) and are regarded as a health food in soups and desserts whereas lotus rhizome is commonly boiled into soup, roasted with honey, fried as chips and pickled with vinegar. Lotus leaves are used to wrap savory rice in Chinese cuisine, they impart the unique fragrance and flavor to the cuisine. Traditionally, lotus seeds are also used in folk medicines to treat tissue inflammation, cancer, diuretics and skin diseases (Mukherjee et al., 2010) while lotus rhizomes are used to treat fever and as a diuretic (Mukherjee et al., 1997). Scientifically, most parts of lotus plant, such as seed (Rai et al., 2006), rhizome (Yang et al., *To whom correspondence should be addressed. changyp@utar.edu.my 2007), stamen (Hyun et al., 2003) and leaves (Huang et al., 2010) exhibited significant antioxidant activities. Lotus plant has metal dominant hypertolerance as it adapts accumulation strategy to accumulate heavy metals from soil. Since the 1900 s, global development in many sectors has accelerated various types of pollution, dramatically. Toxic trace elements are increasing in air, water and soil, as a result of anthropogenic processes. For example, the metal concentration in river water and sediments increased several thousand folds as a result of increased industrial and mining activities (Ashraf et al., 2011). Aquatic plants in freshwater, marine and estuarine systems act as a receptacle for several metals (Prasad and De Oliveira Freitas, 2003). In Malaysia, most of the cultivated lotus is widely grown in ex-tin mining ponds. Perak state in Malaysia was well-known for its tinmining activities between 1890 and 1980 s, with total mining land area of 71,631 hectares (Ang, 1994). Tin reservoir has exhausted and left behind many abandoned ponds which are now being utilized for aquaculture activities including lotus cultivation. Metal contaminants in these ponds can transfer to edible parts of lotus plants, consequently to human food chain. Although lotus plant has been well researched on the bio-

2 462 active compound and beneficial properties (Duan and Jiang, 2008; Kashiwada et al., 2005; Ling et al., 2005) there is little study on lotus plant as a metal transfer media through the food chain. The present study was undertaken to determine the antioxidant properties such as DPPH radical scavenging activity, total phenolic content and tyrosinase inhibition activity and the food safety risk factor such as the concentration of lead and chromium in lotus plant grown in ex-tin mining pond. It is anticipated that the findings may help raise awareness about food safety risk when utilizing abandoned ex-mining land for growing edible health plants. Materials and Methods Plant material preparation and extraction Lotus rhizomes, seed and stem were purchased from local wet market at Kampar, Perak, Malaysia. Fresh plant material, obtained from the market, was washed, cut into small pieces; dried and ground finely (Yang et al., 2007). Moisture content of fresh and ground samples was determined according to standard AOAC method (AOAC 1995). Sample prepared was stored at 20 for later analysis. Ground, dried samples were used directly for determination of ash, chromium and lead. For antioxidant properties determination, a ratio of 1:10 ground sample to methanol were mixed and left overnight at room temperature. Solid-liquid separation was done by vacuum filtration and the filtrate evaporated in a rotary vacuumevaporator (Büchi, Switzerland) at 40 to obtain the sample extract, which was weighed and used later. DPPH radical scavenging activity Radical scavenging activity was evaluated against 1-diphenyl-2-picrylhydrazyl (DPPH) (Huang et al., 2010; Yang et al., 2007). Sample extract of 0.1 ml with various concentrations (0.5 2 mg/ml) was mixed with 2.9 ml of DPPH solution (60 μm). This mixture was shaken vigorously and allowed standing for 30 min in dark. The absorbance of the mixture, A sample was measured at 517 nm against methanol as blank. The radical scavenging activities (%) of test samples were evaluated by comparing with the absorbance of a control (2.9 ml DPPH and 0.1 ml of methanol), A control. The free radical scavenging activity was calculated using Eq. 1. For better comparison of the DPPH scavenging activity, the results obtained from the radical scavenging experiments were also expressed as IC 50 values. IC 50 is the extract concentration at which DPPH radicals were reduced by 50% and calculated from the linear regressional analysis (Khattak et al., 2009). A control-a sample DPPH radical scavenging activity (%) = # 100 (1) A control Tyrosinase inhibition activity Tyrosinase inhibition study was determined based on the method of Masuda et al. (2005). Sample extract of various concentrations (0.5 E.S. Leong et al. 2.0 mg/ml) were mixed with phosphate buffer, L-3,4- dihydroxyphenylalanine (L-dopa) (0.83 mm) and tyrosinase solution (125 units/ml) in a ratio of 1:3:3:1. A mixture without sample extract was regarded as the control. After incubating for 10 minutes at 37 and cooling to 0, the absorbance at 490 nm was measured against a mixture without tyrosinase as blank. Tyrosinase inhibition activity of sample extract was expressed as IC 50 value which is defined as the concentration of sample extract required to inhibit 50% of tyrosinase activity. Total phenolic content Total phenolic content was determined by the Folin-Ciocalteu method (Kähkönen et al., 1999). Aliquots of methanol extract containing 0.6 mg samples extract were mixed with Folin-Ciocalteu reagent (10 times dilution) and sodium carbonate (7.5% w/v) in a ratio of 1:5:4 and kept in the dark for 1 h. Absorbance of the mixture at 765 nm was then recorded. A calibration curve was established with different concentration of tannic acid for estimating the total phenolic content in the samples. Concentration of total phenolic compounds was expressed as mg of tannic acid equivalent per g of sample extract or per 100 g of dry lotus powder (mg TAE/100 g DM). Determination of ash content Ash content was determined gravimetrically (Demirel et al., 2008). Ground sample was weighed into a porcelain crucible. The sample was burned with a Bunsen burner before placing in a Neytech- VULCAN D-1750 model Furnace at 580 overnight to complete the ashing process. Determination of lead and chromium content The ashed sample was digested with concentrated nitric acid and placed in a furnace at 375 for 1 h according to the method of Demirel et al. (2008). Concentration of lead and chromium, in the digested sample was determined at nm and nm respectively by a Perkin-Elmer Analyst 200 model Atomic Absorption Spectrophotometer, based on standard curves constructed from a series of standard solutions ( mg/l). Statistical analysis All analyses were carried out in triplicates and data expressed as means ± standard deviations. One-way analysis of variance (ANOVA) and Duncan s multiple range test (DMRT) were carried out to assess significant differences between means (P < 0.05) using SPSS version Correlations between antioxidant activity and tyrosinase inhibition activity were done utilizing bivariate Pearson procedure. Results and Discussion Evaluation of antioxidant properties DPPH radical scavenging activity of lotus plant extract followed the order of stem < seed < rhizome. The stem exhibited the lowest

3 Antioxidant and Food Risks of Lotus Plant Fig. 1. DPPH radical scavenging activity of lotus seed, rhizome and stem with respective IC 50 values (n = 3). DPPH radical scavenging activity with IC 50 values of 11.7 whereas the rhizome extract showed the highest DPPH radical scavenging activity with IC 50 values of 1.1 (Fig. 1). The IC 50 values are comparable with those reported on lotus rhizome by Khattak et al. (2009). Tyrosinase (polyphenol oxidase), a copper-containing enzyme catalyses the oxidation of hydroxyphenols to their quinone derivatives, which then polymerize spontaneously and initiate enzymatic browning (Shi et al., 2001). The use of natural inhibitors of tyrosinase is stimulated by the need to replace sulfating agents in processed foodstuffs and demand by cosmetic industry for natural whitening agent (Kim et al., 2011). In this study, all extracts of the seed, rhizome and stem of lotus plant exhibited tyrosinase inhibitory effects with IC 50 of 2.2, 1.2 and 2.3 respectively (Fig. 2). The rhizome exhibited the highest tyrosinase inhibitory effect, 2 fold higher as compared to those of seed and stem. Total phenolic content of lotus plant extract followed the order of rhizome < stem < seed, in the range of 2.8 to 12.4 mg TAE/100 g DM or 20.6 to 38.3 mg TAE/g extract, which is comparable to those reported by Yang et al. (2007) and Khattak et al. (2009). Total phenolic content in the stem or rhizome was relatively lower, by 3 to 4 folds as compared to those of seed extract when expressed against the weight of dry matter (Table 1). Stem extract surprisingly showed a higher content of total phenolic compound than rhizome eventhough it exhibited the lowest radical scavenging activity and tyrosinase inhibitory effect. Thus, lotus stem may contain phenolic compounds which possess low DPPHscavenging activity. According to Maisuthisakul and Gordon (2009), different phenolic compounds exhibit different radical-scavenging activity, in which the order of DPPH radical scavenging activity was ellagic acid > gallic acid > tannic acid > arbutin = coumarin among the reference phenolic compounds studied. Correlation between DPPH radical scavenging activity and tyrosinase inhibition activity A positive, high correlation between DPPH radical scavenging activity with tyrosinase inhibition activity was shown for the seed, rhizome and stem of lotus (Fig. 3). This finding is consistent with other report where the DPPH radical scavenging activity increased with the tyrosinase inhibition activity (Maisuthisakul and Gordon, 2009). Ash, lead and chromium content Ash content is directly Sample Table 1. Total phenolic content in lotus plant extracts. Total phenolics (mg TAE/g extracts) Total phenolics (mg TAE/100 g DM) Seed 38.3 a ± a ± 0.2 Rhizome 20.6 b ± b ± 0.2 Stem 25.0 c ± c ± Results were expressed as mean of triplicate measurements ± SD. Means with different superscripts in the columns are significantly (P < 0.05) different. Fig. 2. Tyrosinase inhibition activity of lotus seed, rhizome and stem with respective IC 50 values (n = 3). Fig. 3. Correlation between DPPH radical scavenging activity and percentage of tyrosinase inhibition (n = 12, Pearson correlation coefficient of 0.99 for seed, 0.89 for rhizome and 0.99 for stem at P < 0.01).

4 464 E.S. Leong et al. Table 2. Ash, lead and chromium content of seed, rhizome and stem of lotus plant and safe limits. Content of 1 Sample Ash 2 Lead 3 Chromium 3 Seed a ± a ± a ± 12.7 Rhizome 2.07 b ± b ± b ± 2.4 Stem 1.63 b ± b ± b ± 0.2 Safe limits 4,5,6 Not applicable 0.2, Results were expressed as mean of triplicate measurements ± SD. Means with different superscripts in the columns are significantly (P < 0.05) different. 2 Values in g/kg fresh weight. 3 Values in mg/kg fresh weight. 4,5 Maximum permissible limits for lead by FAO/WHO (2001) and Malaysian Food Act 1983 and Food Regulations 1985 (2004), respectively. 6 Proposed chromium criteria for foodstuffs by Eisler (1986). References Ang, L.H. (1994). Problems and prospects of afforestation on sandy tin tailings in Peninsular Malaysia. J. Trop. Forest Sci., 7, AOAC (1995). Official methods of analysis of AOAC international, 16 ed., Arlington, TX: Association of Official Analytical Chemistry. Ashraf, M.A., Maah, M.J. and Yusoff, I. (2011). Analysis of physio-chemical parameters and distribution of heavy metals in soil and water of ex-mining area of Bestari Jaya, Peninsular Malaysia. Asian J. Chem., 23, Ashraf, M.A., Maah, M.J., Yusoff, I. and Gharibreza, M.M. (2010). Heavy metals accumulation and tolerance in plants growing on ex-mining area, Bestari Jaya, Kuala Selangor, Peninsular Malaysia. ICEEA 2010, Singapore, pp Brand-Williams, W., Cuvelier, M. E. and Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci. Technol., 28, Demirel, S., Tuzen, M., Saracoglu, S. and Soylak, M. (2008). Evalproportional to the amount of inorganic residue in a sample. The higher the ash content means the higher amount of mineral in a particular sample. Lotus seed has the highest mineral content compared with rhizome and stem (Table 2). It has been reported that lotus seed contained high amount of potassium, calcium, magnesium and sodium besides small amount of copper, zinc, manganese and iron (Sridhar and Bhat, 2007) which were beneficial to human health. Lotus seed also showed the highest concentration of chromium (358 mg/kg) and lead (163 mg/kg). It is well known that large amount of heavy metals consumption will lead to serious systemic health problems. Therefore, FAO/WHO Food Standards through Codex Alimentarius and most countries including Malaysia have set maximum permissible limit of various heavy metals in food. Lead in lotus sample studied had exceeded the limits of Codex Alimentarius (FAO/WHO, 2001) and Malaysian Food Act 1983 and Food Regulation Chromium in the seed was slightly higher than the recommended chromium level in food stuffs (Eisler, 1986). These results confirmed previous findings (Ashraf et al., 2010) in which lotus plant has been identified as a good metal dominant hyper-tolerant type of aquatic plant. Lotus can accumulate metals such as lead and chromium in its various parts. Comparatively, lotus plant accumulates higher amount of chromium than lead (Table 2). This is not surprising since some researchers have revealed that chromium is highly mobile and has high plant transfer coefficients (Sridhara Chary et al., 2008). Since concentrations of lead in all parts of lotus plant studied and chromium in lotus seed were noted to be above the critical level and thus a likely threat to consumers. Conclusions Locally grown lotus plant in ex-tin mining pond showed significant antioxidant capacity in terms of DPPH radical scavenging activity, total phenolic content and tyrosinase inhibition activity. Lotus seed extract exhibited the highest total phenolic content whereas lotus rhizome exerted the highest tyrosinase inhibition activity. Lotus plant studied also contains significant amount of heavy metal such as lead which exceeded the maximum permissible limit despite its significant antioxidative properties. Metal contamination assessments in addition to remediation on ex-tin mining site should be carried out prior to agricultural activities; to minimize heavy metal transferring to human food chain. Acknowledgements This study was supported by Universiti Tunku Abdul Rahman, Malaysia. Authors are thankful to Dr Tan Chew Khun, Faculty of Engineering and Green Technology, Universiti Tunku Abdul Rahman, Malaysia and the reviewers for their critical inputs on this manuscript.

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