Bot. Bull. Acad. Sin. (2005) 46: 183-188

LIN et al. — Antioxidant activities of mucilages from different Taiwanese yam cultivars

Antioxidant activities of mucilages from different Taiwanese yam cultivars

Shyr-Yi LIN^1,2, Hao-Yu LIU³, Yeh-Lin LU³, and Wen-Chi HOU^3,*

¹Department of Internal Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan

²Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan

³Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan 110

(Received January 31, 2005; Accepted April 7, 2005)

Abstract. The antioxident effects of crude mucilages (CM) and partially purified mucilages (PPM) from three different Taiwanese yam cultivars—including Dioscorea alata L. cv. Tainong 1 (TN1), Dioscorea alata L. cv. Tainong 2 (TN2), and D. alata L. var. purpurea (Roxb.) Ming-Jen (MJ)—were evaluated, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, and superoxide radical scavenging activities Electron spin resonance (ESR) spectrometry was used to measure hydroxyl radical scavenging activities. The IC₅₀ stands for the concentration required for 50% scavenging activity. The IC₅₀ of CM and PPM against DPPH radical was 0.329, 0.279; 0.547, 0.653; and 0.847, 0.631 mg/ml, respectively, for TN1, TN2 and MJ. The IC₅₀ of CM and PPM against hydroxyl radical by spectrophotometry was 0.668, 1.146; 1.461, 1.096; and 0.946, 1.554 mg/ml, respectively, for TN1, TN2 and MJ. The IC₅₀ of CM and PPM against superoxide radical was 0.802, 0.368; 0.681, 0.258; and 0.086, 0.148 mg/ml, respectively, for TN1, TN2 and MJ. Using ESR to detect hydroxyl radicals, the IC₅₀ of PPM against hydroxyl radical was 0.083, 0.47, and 0.004 mg/ml, respectively, for TN1, TN2 and MJ. The results demonstrated that different cultivars of yams exhibited different antioxidant ability, and the purification process was able to partially increase the antioxidant activity of the mucilage polysaccharide. Taken together, these results suggest that mucilage polysaccharides of the yam tuber might play an important role on antiradicals and antioxidants.

Keywords: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical; Electron spin resonance (ESR); Hydroxyl radical; Mucilage; Superoxide radical; Yam.

Introduction

Active (or reactive) oxygen species and free radical-mediated reactions are involved in degenerative or pathological processes such as aging (Harman, 1995), cancer, coronary heart disease and Alzheimer's disease (Ames, 1983; Smith et al., 1996; Diaz et al., 1997). Meanwhile, many epidemiological results support an association between a diet rich in fresh fruit and vegetables and a decrease in the risk of cardiovascular diseases and certain forms of cancer (Salah et al., 1995) in humans. Several reports concern the antioxidant activities of the natural compounds in fruit and vegetables, such as phenolic compounds (Rice-Evans et al., 1997), anthocyanin (Espin et al., 2000), echinacoside in Echinaceae root (Hu and Kitts, 2000), methanolic and hot-water extracts of Liriope spicata L. (Hou et al., 2004), water and ethanolic extracts of different sweet potato organs (Huang et al., 2004), the storage proteins of sweet potato root (Hou et al., 2001a), yam tuber (Hou et al., 2001b), and potato tuber (Liu et al., 2003). In cells, certain metabolic pathways normally degrade free radicals. If the rate of free radical generation exceeds that of degradation under environmental stresses, cells suffer

oxidative stress. Two distinct pathways, nonenzymatic and enzymatic, were found in plant cells as routes of free radical scavengers. The former included ascorbate (Njus and Kelley, 1993), chlorogenic acids (Kono et al., 1998), and vitamin E (Halliwell, 1999); the latter included different forms of superoxide dismutase to metabolize superoxide free radical to hydrogen peroxide (Bowler et al., 1992; Lin et al., 1993; Hou et al., 2003). The hydrogen peroxide produced was further metabolized either by catalase or different forms of peroxidase such as glutathione peroxidase (EC 1.11.1.9).

Yam (Dioscorea species) is a member of the monocotyledonous family Dioscoreaceae and is a staple food in West Africa, Southeast Asia, and the Caribbean (Akoruda, 1984). Yam has been recognized as an herb since the dried tuber slices were first used as Chinese medicine. The tuber storage protein of yam, dioscorin, has exhibited carbonic anhydrase, trypsin inhibitor activities (Hou et al., 1999a) and both dehydroascorbate reductase and monodehydroascorbate reductase activities (Hou et al., 1999b). Yam tuber contains mucilages which were reported to be a mannan-protein complex (Misaki et al., 1972; Tsai and Tsai, 1984). Recently, we reported that yam tuber mucilage exhibited angiotensin converting enzyme inhibitory activities (Lee et al., 2003). In this work crude mucilages (CM) and partially purified mucilages (PPM) from three different Taiwanese yam cultivars—including Dioscorea

*Corresponding author. Fax: 886-2-2378-0134; E-mail: wchou@tmu.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

alata L. cv. Tainong 1 (TN1), Dioscorea alata L. cv.Tainong 2 (TN2), and D. alata L. var. purpurea (Roxb.) Ming-Jen (MJ)—were used to evaluate the antioxidant effects of scavenging DPPH radical, hydroxyl radical, and superoxide radical. We used spectrophotometry, and hydroxyl radical scavenging activity assay was done by electron spin resonance (ESR) spectrometry.

Materials and Methods

Materials

1,1-diphenyl-2-picrylhydrazyl (DPPH), NADH, 5,5-dimethyl-1- pyrroline-N-oxide (DMPO), ferrous sulfate, and phenazine methosulfate (PMS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide (33%) was from Wako Pure Chemical Industry (Osaka, Japan). Other chemicals and reagents were from Sigma Chemical Co. (St. Louis, MO, USA).

Extraction and Purificaction of Mucilage from Yam Tuber

Fresh yam tubers of TN1, TN2, and MJ were purchased from a wholesaler. After washing and peeling, the tubers were cut into strips for mucilage extractions and purifications according to the methods of Lee et al. (2003) with some modifications. Yam tuber was homogenized with four volumes (W/V) of 50 mM Tris-HCl buffer (pH 8.3) containing 1% vitamin C. After centrifugation at 14,000 g for 30 min, the supernatants were mixed with isopropanol to a final concentration of 70%, and stirred quickly at 4ºC overnight. The precipitates were filtrated and dehydrated with 100% isopropanol, then rinsed with acetone. After drying at 40ºC in an oven, the crude mucilage (CM) was ground and collected for further purifications by both SDS and heating procedures. About 1.0 g CM powder was dissloved in 200 ml distilled water and kept in a 50ºC water bath. Forty ml of 5% SDS solution (dissloved in 45% ethanol) was added to the CM solution. The mixture was kept with gentle stirring at 50ºC for 30 min, then, at room temperature for another 2 h. After that, the mucilage solution was placed in an ice bath to quickly reduce the temperature and precipitate the SDS-protein complex. After centrifugation at 14,000 g at 0ºC for 30 min, the supernatants were precipitated with isopropanol and dried at 40ºC in an oven as described earlier. The mucilage was again ground, dissolved, and then heated in boiling water for 20 min. After centrifugation at 14,000 g at 0ºC for 30 min, the supernatants were mixed with isopropanol to a final concentration of 70%. The partially purified mucilage (PPM) was filtrated, dehydrated, rinsed with acetone, dried, and then collected for further uses.

Scavenging Activitiy Against 1,1-Dipheny-2-Picrylhydrazyl (DPPH) Radical Analyzed by Spectrophotometry

The scavenging activity of CM and PPM from TN1, TN2, and MJ cultivars against DPPH radical was measured

according to the method of Hou et al. (2001a, b). Each 0.3 ml of CM (0.25, 0.5, 1.0 and 1.5 mg/ml) and PPM (0.1, 0.15, 0.3, 0.5 and 1.0 mg/ml) solution was added to 0.1 ml of 1 M Tris-HCl buffer (pH 7.9), and then mixed with 0.6 ml of 100 µM DPPH in methanol for 20 min under light protection at room temperature. After brief centrifugation at 12,000 g for 10 min, the absorbance at 517 nm was measured. Deionized water was used as a blank. The scavenging activity of DPPH radicals (%) was calculated following the equation: (A517_blank - A517_sample) ÷ A517_blank × 100%. The IC₅₀ stands for the concentration required for 50% scavenging activity and was calculated from the above equation.

Scavenging Activity of CM and PPM Against Metal Ion-Dependent Hydroxyl Radicals

The hydroxyl radical was determined by the deoxyribose method (Halliwell et al., 1987). Every 0.5 ml sample containing different amounts of CM (0.375 , 0.75, and 1.5 mg/ml) and PPM (0.4, 0.8, and 1.6 mg/ml) from TN1, TN2, and MJ cultivars were added to 1.0 ml solution of 20 mM potassium phosphate buffer (pH 7.4), 2.8 mM 2-deoxy-ribose, 104 µM EDTA, 100 µM FeCl₃, 100 µM ascorbate, and 1 mM hydrogen peroxide. The mixtures were incubated for 1 h at 37ºC. After incubation, an equal volume of 0.5% thiobarbituric acid in 10% trichloroacetic acid was added, and the mixtures were boiled at 100ºC for 15 min. Deionized water was used as a blank experiment. The absorbance at 532 nm was measured. The scavenging activity of hydroxyl radicals (%) was calculated with the equation: (A532_blank - A532_sample) ÷ A532_blank × 100%. The IC₅₀ stands for the concentration required for 50% scavenging activity and was calculated from the above equation.

Scavenging Activity Against Superoxide Radicals Analyzed by Spectrophotometry

The superoxide radical was generated by the PMS-NADH system (Lai et al., 2001). Every 0.2 ml sample containing different amounts of CM (0.125, 0.25, 0.5, and 1.0 mg/ml) and PPM (0.125, 0.5, 1.0 mg/ml) solution from TN1, TN2, and MJ cultivars was added in sequence to 0.2 ml of 630 µM nitroblue tetrazolium, 0.2 ml of 33 µM PMS, and 0.2 ml of 156 µM NADH in 100 mM phosphate buffer (pH 7.4). Means of triplicates were measured. Deionized water was used as a blank experiment. The changes of absorbance at 560 nm were recorded during 1 min and expressed as DA560nm/min. The scavenging activity against superoxide radicals was calculated as follows: (DA560 nm/min_blank - DA560 nm/min_sample) ÷ DA560 nm/min_blank × 100%. The IC₅₀ stands for the concentration of 50% scavenging activity.

Scavenging Activities Against Hydroxyl Radical by Electron Spin Resonance Spectrometry

The hydroxyl radical was generated by Fenton reaction according to the method of Kohno et al. (1991). The total 500-µl mixture contained different concentrations of PPM solution of TN1 (0.031, 0.062, 0.1, and 0.2 mg/ml), TN2

LIN et al. — Antioxidant activities of mucilages from different Taiwanese yam cultivars

(0.12, 0.25, 0.5, and 1 mg/ml), and MJ (0.0004, 0.004, and 0.007 mg/ml) cultivars, 5 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and 0.05 mM ferrous sulfate. After mixing, the solution was transferred to an ESR quartz cell and placed at the cavity of the ESR spectrometer, and then hydrogen peroxide was added to a final concentration of 0.25 mM. Deionized water was used instead of sample solution for blank experiments. After 40 s, the relative intensity of the signal of the DMPO-OH spin adducts was measured. All ESR spectra were recorded at the ambient temperature (298 K) on a Bruker EMX-6/1 EPR spectrometer equipped with WIN-EPR SimFonia software, Version 1.2. The conditions of ESR spectrometry were as follows: center field, 345.4 (5.0 mT; microwave power, 8 mW (9.416 GHz); modulation amplitude, 5 G; modulation frequency, 100 kHz; time constant, 0.6 s; scan time, 1.5 min.

Results and Discussion

Yam (Dioscorea species) is a member of the monocotyledonous family Dioscoreaceae and is a staple food in West Africa, Southeast Asia, and the Caribbean (Akoruda, 1984). Yam has been recognized as an herb since the dried tuber slices were first used as Chinese medicine. In this study, three native cultivars of Taiwanese yam were used for mucilage isolation and purification, and then for antioxidant activity assay.

DPPH radicals were widely used in the model system to investigate the scavenging activities of several natural compounds. When DPPH radical was scavenged, the color of the reaction mixture changed from purple to yellow with absorbance decreasing at wavelength 517 nm. Figure 1A shows the scavenging activity against DPPH radicals of CM from TN1, TN2, and MJ yam cultivars. Dose-dependent DPPH radical scavenging activities of CM were found in three native yam cultivars. The order of DPPH scavenging activity was TN1 > TN2 > MJ. The IC₅₀ of CM against DPPH radical were 0.329, 0.547, and 0.847 mg/ml, respectively, for TN1, TN2 and MJ cultivars. After being purified with SDS and heated in boiling water, the PPM of three yam cultivars was again assayed for antioxidant activity. Figure 1B shows the scavenging activity against DPPH radicals of PPM from TN1, TN2 ,and MJ cultivars. It was found that the DPPH radical scavenging activities of PPM were better than those of CM from three native yams. The IC₅₀ of PPM against DPPH radical were 0.279, 0.653, and 0.631 mg/ml, respectively, for TN1, TN2 and MJ cultivars. Our previous study reported (Hou et al., 2002) that PPM from Japanese yam also exhibited DPPH scavenging activity, and the IC₅₀ of PPM against DPPH radical was 0.86 mg/ml, which was similar to what Lai et al. (2001) reported for Hsian-tsao leaf gum and higher than those of PPM from TN1, TN2, and MJ cultivars.

Figure 2 shows the scavenging activity against hydroxyl radical from CM (A) and PPM (B) of TN1, TN2, and MJ yam cultivars. As in Figure 1, dose-dependent hydroxyl radical scavenging activities of CM were found in three native yam cultivars. The order of hydroxyl radical scav

enging activity was TN1 > MJ > TN2 (Figure 2A). The IC₅₀ of CM against hydroxyl radical was 0.668, 1.461, and 0.946 mg/ml, respectively, for TN1, TN2 and MJ cultivars. Figure 2B shows the scavenging activity against hydroxyl radicals of PPM from TN1, TN2 ,and MJ cultivars. The hydroxyl radical scavenging activities of PPM were lower than those of CM from three native yams. The IC₅₀ of PPM against hydroxyl radical was 1.146, 1.096, and 1.554 mg/ml, respectively, for TN1, TN2 and MJ cultivars. Previously, we reported that the tuber storage protein, dioscorin, exhibited hydroxyl radical scavenging activity (Hou et al., 2001b). The dioscorin should have been removed during SDS and heating treatments, which would have resulted in less hydroxyl radical scavenging activity of PPM in the three native yam cultivars.

Figure 3 shows the scavenging activity against superoxide radical from CM (A) and PPM (B) of TN1, TN2, and MJ yam cultivars. As with Figures 1 and 2, dose-dependent superoxide radical scavenging activities of CM were found in three native yam cultivars. The order of superoxide radical scavenging activity was MJ > TN2 > TN1 (Figure 3A). The IC₅₀ of CM against superoxide radical was 0.802,

Figure 1. The scavenging activity of crude mucilage (CM) (A) and partially purified mucilage (PPM) (B) of TN1, TN2, and MJ yam cultivars against DPPH radical. Means of triplicates were measured. Deionized water was used as a blank experiment. The scavenging activity of DPPH radical (%) was calculated according to the following equation: (A517_blank - A517_sample) ÷ A517_blank × 100%.

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

Figure 2. The scavenging activity of crude mucilage (CM) (A) and partially purified mucilage (PPM) (B) of TN1, TN2, and MJ yam cultivars against hydroxyl radical. Means of triplicates were measured. Deionized water was used as a blank experiment. The absorbance at 532 nm was measured. The scavenging activity of hydroxyl radicals (%) was calculated with the equation: (A532_blank - A532_sample) ÷ A532_blank × 100%.

Figure 3. The scavenging activity of crude mucilage (CM) (A) and partially purified mucilage (PPM) (B) of TN1, TN2, and MJ yam cultivars against superoxide radical. Means of triplicates were measured. Deionized water was used as a blank experiment. The changes of absorbance at 560 nm were recorded during 1 min and expressed as DA560 nm/min. The scavenging activity of the superoxide radical was calculated as follows: (DA560 nm/min_blank - DA560 nm/min_sample) ÷ DA560 nm/min_blank × 100%.

0.681, and 0.086 mg/ml, respectively, for TN1, TN2 and MJ cultivars.

The hydroxyl radical was generated by Fenton reaction and was trapped by DMPO to form DMPO-OH adduct. The intensities of DMPO-OH spin signal in ESR spectrometry were used to evaluate the scavenging activity of the PPM of TN1, TN2, and MJ yam cultivars against hydroxyl radical. Figure 4 shows scavenging activities against hydroxyl radicals of (A) TN1 (0.031, 0.062, 0.1, and 0.2 mg/ml), (B) TN2 (0.12, 0.25, 0.5, and 1 mg/ml), and (C) MJ (0.0004, 0.004, and 0.007 mg/ml) cultivars. From the results of Figure 4A, the scavenging activities against hydroxyl radical were 1, 26, 69.6, and 81% at 0.031, 0.062, 0.1, and 0.2 mg/ml, respectively, of PPM from TN1 cultivars. From the results of Figure 4B, the scavenging activities against hydroxyl radical were 13, 31.5, 51.9, and 72.2% at 0.12, 0.25, 0.5, and 1 mg/ml, respectively, of PPM from TN2 cultivars. From the results of Figure 4C, the scavenging activities against hydroxyl radical were 5.5, 50, and 51.8%

at 0.0004, 0.004, and 0.007 mg/ml, respectively, of PPM from MJ cultivars. Using ESR to detect hydroxyl radicals, the IC₅₀ of PPM against hydroxyl radical were 0.083, 0.47, and 0.004 mg/ml, respectively, for TN1, TN2 and MJ yam cultivars.

In conclusion, mucilages from three Taiwanese yams exhibited different antioxidant activities against DPPH radicals (Figure 1), hydroxyl radicals (Figure 2, Figure 4), and superoxide radicals (Figure 3), and the purification process was able to partially increase the antioxidant activity of the mucilage polysaccharide. Table 1 shows the comparison of the antioxidant activity (IC₅₀) of mucilages from TN1, TN2, and MJ yam cultivars before (crude mucilage, CM) and after purification (partially purified mucilage, PPM) against DPPH, hydroxyl, and superoxide radicals. Taken together, these results suggest that mucilage polysaccharides of yam tuber might be important antiradicals and antioxidants.

LIN et al. — Antioxidant activities of mucilages from different Taiwanese yam cultivars

A

Acknowledgments. The authors want to thank the National Science Council, Republic of China for its financial support (NSC93-2313-B038-001).

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Figure 4. The scavenging activity of partially purified mucilage (PPM) of (A) TN1 (0.031, 0.062, 0.1, and 0.2 mg/ml), (B) TN2 (0.12, 0.25, 0.5, and 1 mg/ml), and (C) MJ (0.0004, 0.004, and 0.007 mg/ml) cultivars against hydroxyl radical. The signal intensities of DMPO-OH adduct were determined by electron spin resonance spectrometry. The scavenging activity against hydroxyl radical was also shown in the figure.

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