Food Chemistry 124 (2011) 476–480
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An antioxidant protein in Curcuma comosa Roxb. Rhizomes Apaporn Boonmee a, Chantragan Srisomsap b, Aphichart Karnchanatat c, Polkit Sangvanich a,* a
Research Center for Bioorganic Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand c The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand b
a r t i c l e
i n f o
Article history: Received 27 November 2009 Received in revised form 10 May 2010 Accepted 15 June 2010
Keywords: Curcuma comosa Roxb. Antioxidant Superoxide dismutase Plant protein
a b s t r a c t Curcuma comosa Roxb. is an indigenous Thai herb which is usually used as a food ingredient but it is also used in traditional folk medicine for the treatment of uterine inflammation. The crude protein extract from the rhizomes of this plant was found to possess free radical scavenging capacity, as detected by the DPPH assay. This antioxidant activity was purified by DEAE anion exchange chromatography to a fraction (called IE-1) that was comprised of a single main protein band of 18 kDa (UB-DEAE), as determined by SDS–PAGE resolution with Coomassie blue staining, and had a specific activity of 193.8 U/mg. In-gel trypsin digestion of the SDS–PAGE resolved UB-DEAE band followed by liquid chromatography–tandem mass spectrometry produced three reliably sequenced peptides, which all were found to very likely be fragments of a superoxide dismutase homologue (SOD, EC 1.15.1.1), an antioxidant enzyme that has been found in several plants. In support of this notion, the IE-1 fraction was found to yield positive results with the riboflavin–nitroblue tetrazolium (NBT) assay, a standard test for SOD-like activity. Together, these data then support the existence of an SOD homologue antioxidant protein in the rhizomes of C. comosa as a contributing agent to the total observed antioxidant activity. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Several plants in the Curcuma genus (Zingiberales: Zingiberaceae) have been widely used as food, food flavouring and spice agents and traditional folk medicine in many countries of Asia, including Thailand. Curcuma comosa, commonly known as ‘‘Waan Chak Mod Look” in Thai language, has been used as a food ingredient, to medically treat postpartum uterine bleeding and as an aromatic stomachic. This plant, and especially the rhizomes, has been found to harbour various biological properties, such as oestrogenic (Winuthayanon, Piyachaturawat et al., 2009; Winuthayanon, Suksen et al., 2009), nematocidal (Jurgens et al., 1994), anti-inflammatory (Jantaratnotai, Utaisincharoen, Piyachaturawat, Chongthammakun, & Sanvarinda, 2006), and choloretic (Piyachaturawat, Charoenpiboonsin, Toskulkao, & Suksamrarn, 1999) activities. In the normal body’s functions, free radicals are by-products of specific oxidation reactions, often for the elimination of pathogens or infected cells. Excess of these highly reactive species can initiate chain reactions that damage cells and, if not controlled, cause many diseases. Thus, although their generation is important, for example in control of pathogens, so is their localised controlled destruction to protect unwanted damage to healthy host cells. Substances which can stop these chain reactions by removing free rad* Corresponding author. E-mail address:
[email protected] (P. Sangvanich). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.06.057
icals are called ‘‘antioxidants”. Anti-oxidation substances in nature can be divided into two groups (Huang, Ou, & Prior, 2005); (i) the enzymatic group, for example superoxide dismutase (SOD), glutathione peroxidase and catalase, and (ii) the non-enzymatic antioxidants, for example carotenoids, ascorbic acid, a-tocopherol, glutathione and phenolics. With the growing medical interest in natural antioxidants, it is of no surprise that antioxidant proteins in plants have gained attention and reports of their existence and bioactivities have correspondingly increased as well (Arcan & Yemenicioglu, 2007; Ningappa & Srinivas, 2008; Rios-Gonzalez, Erdei, & Lips, 2002; Sarkar, Kinter, Mazumder, & Sil, 2009; Sivapriya & Leela, 2007). Recently, Curcuma longa L., or turmeric, was found to have a potential antioxidant protein, b-turmerine (Smitha, Dhananjaya, Dinesha, & Srinivas, 2009). Although an antioxidant activity was also been reported in the related Curcuma comosa (Niumsakul, Hirunsaree, Wattanapitayakul, Junsuwanitch & Prapanupun, 2007) this was found to be due to a small nonprotein molecule, 4,6-dihydroxy-2-O-(b-D-glucopyranosyl) acetophenone, and showed not only a powerful antioxidant activity but was also cytotoxic to HeLa cells in tissue culture. However, to our knowledge there is no previous report on any antioxidant proteins from C. comosa, yet this remains of interest due to the folklore medicinal usage. Consequently, this research aimed to find if any of the antioxidant activity in C. comosa rhizomes was attributed to protein components, by screening for antioxidant activity using the DPPH assay.
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2. Materials and methods 2.1. Reagents 2,2-Diphenyl-1-picryhydrazyl (DPPH), nitroblue tetrazolium (NBT) and vitamin E were purchased from Sigma Chemicals Co. (USA). The reagents used in SDS–PAGE were obtained from Plusone Pharmacia Biotech (Sweden), except the low molecular weight calibration kit, used as standard molecular weight marker proteins, which was purchased from Amersham Pharmacia Biotech (UK). All other biochemicals and chemicals used in the investigation were of analytical grade.
100
A280 (mAU)
80
80
IE-2
60
One kilogram of C. comosa rhizomes, purchased from a local market in Bangkok, Thailand. A voucher specimen (BKF. No. 97298) is deposited at The Forest Herbarium (BKF), Royal Forest Department, Bangkok, Thailand. The rhizomes were peeled, minced into small cubes, and blended and extracted overnight at 4 °C in 4 L of extraction buffer (20 mM Tris–HCl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 and 0.15 M NaCl). After filtration through cheesecloth, the crude extract was centrifuged at 15,000g for 20 min. The supernatant was harvested, ammonium sulphate added to a final concentration of 90% saturation and left overnight at 4 °C. The precipitate suspension was collected after centrifugation as above but for 30 min, dissolved in deionized water and then dialysed against 20 mM Tris–HCl pH 7.4 (buffer A). The solution (5 mL) was applied to a DEAE-cellulose anion exchange column (1.6 15 cm) installed in an AKTA prime instrument (GE Healthcare, Uppsala, Sweden) and equilibrated with buffer A. The bound fraction was eluted with a stepwise gradient (see Fig. 1 for profile) formed by the addition of 0%, 20%, 50%, 80% and 100% (v/v) of buffer B (20 mM Tris–HCl pH 7.4 containing 0.5 M NaCl), and collecting 5 ml fractions. The collected fractions were dialysed against water and analysed for (i) antioxidant activity, as detailed below, and (ii) protein concentration using the Bradford assay (Bradford, 1976), with bovine serum albumin (concentrations between 0 and 20 lg/ml) as the standard.
60
IE-1 40
40
20
IE-4
IE-5
0
20
0 0
50
100
150
200
250
Elution volumn (ml) Fig. 1. DEAE anion exchange chromatography of the crude protein extract from the rhizomes of Curcuma comosa Roxb. Bound proteins were eluted with a stepwise gradient of NaCl as detailed in the methods. The cumulative total amount of boundproteins eluted after each fraction, as a per cent of the total initial DEAE-bound amount (% B), is shown as the dash line, whilst the absorbance at 280 nm of the eluate at each fraction is shown as the solid line.
2.3. Measurement of free radical scavenging capacity by DPPH assay The antioxidant activity of each of the fractions was determined using DPPH as reported (Udenigwe, Lu, Han, Hou, & Aluko, 2009). The different concentrations of the protein sample (12.5–200 lg/ ml; total volume of 40 lL) in 96-well plates were mixed with 160 lL of 100 mM DPPH in methanol and then incubated in the dark for 20 min at room temperature prior to reading the absorbance at 517 nm in a micro plate reader. A negative control, containing water instead of the sample, positive controls, containing either 1.135 mM ascorbic acid or 0.464 mM vitamin E in place of the protein sample, and blank samples, using the same volume of methanol only in place of the DPPH solution in methanol, were all evaluated at the same time per micro titre plate. The percentage of radical scavenging was calculated as follows;
% radical scavenging ¼ 2.2. Protein extraction and isolation
100
IE-3
%B
Within the spectrum of antioxidant proteins is superoxide dismutase (SOD), one of antioxidant enzyme systems which protects cells against toxic oxygen free radicals produced during normal metabolism and after oxidative insult (Sun, 1990), by catalysing the dismutation of superoxide radicals to oxygen and hydrogen peroxides. SODs are widely used in medicine, pharmaceutical (Fattman, Schaefer, & Oury, 2003; Noor, Mittal, & Iqbal, 2002) and the food industry (Lingnert, Aakesson, & Eriksson, 2002; Nice & Robinson, 1992), and so of some interest. They have been isolated from various plants, such as citrus (Almansa, Palma, Yanez, del Rio, & Sevilla, 1991), tobacco (Sheng et al., 2004), tea (Vyas & Kumar, 2005), garlic (Hadji et al., 2007), pea (Nice, Robinson, & Holden, 1995) and rice (Padiglia, Medda, Cruciani, Lorrai, & Floris, 1996). Recently, SOD enzymes being have been reported in two members of the Zingeberaceae plant family, that is in the leaves and rhizomes of C. longa L., which interestingly this SOD enzyme was found to be stable to high temperatures and harsh conditions (Kochhar & Kochhar, 2008), and in Curcuma zedoaria Roscoe. (Loc et al., 2008). Thus, when one of the purified protein fractions with antioxidant activity (IE-1) was resolved by reducing SDS–PAGE and provisionally identified by in-gel tryptic digestion followed by peptide analysis using tandem mass spectrometry as likely containing a SOD homologue, this fraction was also assayed for superoxide dismuatase (SOD) enzyme activity.
ðAc AcbÞ ðAs AsbÞ 100 ðAc AcbÞ
where Ac is the absorbance of water plus DPPH (in methanol), Acb is the absorbance of the blank (water plus methanol without DPPH), As is the absorbance of the sample plus DPPH (in methanol) and Asb is the absorbance of the sample plus methanol without DPPH. Different sample concentrations were used in order to obtain calibration curves and to calculate the EC50 values (EC50: concentration required to obtain a 50% radical scavenging activity). 2.4. Measurement of superoxide dismutase (SOD) activity To determine the SOD activity, the riboflavin-NBT assay was adapted from Lai (Lai, Chang, & Chang, 2008). The test sample (0.1 mL) at different protein concentrations were first mixed with 2.75 mL of 67 mM phosphate buffer (pH 7.8) containing 0.01 M EDTA and 0.1 mL of 1.5 mM NBT. After incubation at 37 °C for 5 min, 0.05 mL of 1.2 mM riboflavin was added. The reaction mixture was moved to a foil-lined box and illuminated with a 25 W light tube for 15 min. The inhibition of NBT reduction was determined at measuring the absorbance at 560 nm by a micro titre plate reader. A negative control (water instead of the sample), positive control, and blank (addition of water instead of the riboflavin solution) were evaluated at the same time per micro titre plate. In addition, a set of standards were evaluated and these were then used to determine the SOD activity in each test sample where SOD activity was defined as that 1 unit is the amount of enzyme that
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provides a 50% inhibition of the riboflavin-mediated reduction of NBT. 2.5. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) To visualise proteins during the purification steps, reducing SDS–PAGE was performed following the procedure of Lammeli (Laemmli, 1970). Firstly, the protein samples were treated with reducing sample buffer (62.5 mM Tris–HCl pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 14.4 mM 2-mercaptoethanol), followed by boiling for 5 min. After centrifugation as above, 25 lL of the supernatant was applied onto a 15% (w/v) acrylamide running gel. Electrophoresis was conducted at a constant current (20 mA) in 50 mM Tris–glycine–SDS (pH 8.3) running buffer for 2 h. Known protein markers (Amersham Pharmacia Biotech) were used to determine the apparent molecular mass of the proteins, and Coomassie brilliant blue G-250 staining was used to visualise the protein bands. 2.6. Protein characterisation by tandem mass spectrometry After electrophoresis, the principal protein band was excised (see Fig. 2) and in-gel digested with trypsin as reported (Shevchenko, Tomas, Havilis, Olsen, & Mann, 2006). The tryptic peptides were resuspended with 0.1% (v/v) formic acid and then analysed by liquid chromatography coupled with tandem mass spectrometry (LC/MS-MS; model Finnigan LTQ Linear Ion Trap Mass Spectrometer). For identification of the proteins, the tandem mass spectra were interpreted and searched in the MS BLAST database (http:// dove.embl-heidelberg.de/Blast2/msblast.html). HSP (High scoring
Fig. 2. SDS–PAGE of the crude protein and the DEAE-unbound fraction (UB-DEAE), from the rhizomes of C. comosa. The UB-DEAE sample is from the IE-1 eluate of Fig. 1. Lane 1 (Marker) is 10 lg of the low protein MW markers from Amersham Pharmacia Biotech, lane 2 (Crude) is 40 lg of the crude rhizome protein extract after 90% saturation ammonium sulphate precipitation, lane 3 is 15 lg of the unbound DEAE fraction (IE-1), lane 4 is 60 lg of 100 mM NaCl eluted fraction (IE-2) and lane 5 is 90 lg of the 250 mM NaCl eluted fraction (IE-3).
pairs) is a region of high local sequence similarity between the protein in a database and the peptide in the query. MS BLAST provides a statistically significant match when the total HSP score is higher than the threshold value.
3. Results and discussion C. comosa is a herb used in folklore medicine and from which many biologically active small molecules have been reported along with biological functions of the plant extracts, especially the rhizomes, yet no biologically active protein from this plant has been reported to date. Rhizomes from C. comosa were extracted in an aqueous buffer suitable for proteins and then precipitated with 90% saturation ammonium sulphate to concentrate the protein and eliminate small chemical impurities which may otherwise interfere with the antioxidant assays. A dark brown crude protein preparation was obtained from the precipitation step and this was dialysed against deionized water and the antioxidant activity measured by the DPPH assay. The crude protein preparation was found to contain antioxidant activity with a specific activity of 125 and 120 U/mg for extract and ammonium sulphate precipitated fraction, respectively. This crude protein preparation was then subjected to further purification by fractionation through a DEAE anion exchange resin column. The proteins were separated into three main peaks in chromatogram, one unbound (IE-1) and two main DEAE-anion resin bound peaks that were eluted with 100 (IE-2) and 250 (IE-3) mM NaCl, respectively. The two minor peaks (IE-4 and IE-5), eluted with 400 and 500 mM NaCl, respectively (Fig. 1) contained insufficient protein for analysis. Each fraction was collected and evaluated for antioxidant activity. Antioxidant activity, as defined by the DPPH assay, was detected in all three main protein fractions (IE1–3), with the highest total activity found in the 250 mM NaCl eluted fraction (IE-3), some 4.9- and 6.4-fold higher than in the IE-1 and IE-2 fractions, respectively. However, the unbound fraction (IE-1) had the highest specific antioxidant activity when compared with the other two fractions, being some 2.7- and 10-fold higher than that in the IE3 and IE-2 fractions, respectively (Table 1). Thus, in this report we focused on further characterisation of the IE-1 fraction. Reducing SDS–PAGE was used to study the apparent purity (homogeneity) of the protein composition of the IE-1 fraction, within the limitations of resolution provided by one dimensional PAGE and coomassie staining (required, as opposed to silver staining, for subsequent tryptic peptide analysis), as well as its apparent molecular size. From Fig. 2, the IE-1 fraction was comprised of only one detected (principal) band (UB-DEAE) with an apparent molecular weight of approximately 18 kDa. To identify the protein(s) in the UB-DEAE band, assuming it is responsible for the antioxidant activity seen in the IE-1 fraction, the band in the gel was excised and in-gel digested by trypsin. The resultant mixture of eluted tryptic peptides was evaluated with tandem mass spectrometry to yield tandem mass spectra (data not shown), from which the amino acid sequence of each principal peptide was deduced. The sequence of three peptides were resolved reliably well enough to be used to compare against existing known proteins with the MS-Blast against the NCBI database (Fig. 3). All three peptides matched significantly with the antioxidant SOD homologous enzyme from 16 different plants (Table 2), that is each peptide had a total HSP score of more than the threshold score (HSP = 137), and matched to the same 152 bp fragment of the SOD homologues at positions 4–15, 115–134 and 143–151 with a high degree of sequence conservation within these 16 SOD homologues, but not in other non-SOD protein domains (BLAST searches of the NCBI GenBank, not shown), suggesting the, or one of the protein(s) in the UB-DEAE band is likely to be a SOD homologue, consistent with the antioxidant activity of the IE-1 fraction.
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A. Boonmee et al. / Food Chemistry 124 (2011) 476–480 Table 1 Enrichment of the anti-oxidative protein from C. comosa rhizomes. Purification step Crude extract 90% (NH4)2SO4 precipitation IE-1 IE-2 IE-3
Total protein (mg) 2125.0 1139.0 49.3 385.6 660.4
Total activity (U) 5
2.656 10 1.366 105 9.56 103 7.40 103 4.74 104
Specific activity (U/mg)
Protein yield/activity yield (%)
Purification (fold)
125.0 119.9 193.8 19.2 71.8
100/100 53.6/51.4 2.3/3.8 18.1/2.91 31.1/18.7
1.00 0.96 1.55 0.15 0.57
IE-1–IE-3 refer to the unbound (IE-1) and DEAE anion exchange resin bound fractions released with 100 mM NaCl (IE-2) and 250 mM NaCl (IE-3), as shown in Fig. 1. Too little protein was present in peaks IE-4 and IE-5 for analysis.
Fig. 3. Comparison of the amino acid sequences of the three tryptic peptides from the 20 kDa UB-DEAE protein from C. comosa rhizomes with that of SODs from various plants. The 16 plant SOD partial sequences shown are from the same SODs as those with high scoring HSP hits (Table 2), with the accession codes given in Table 2.
Table 2 Potential identification of the purified protein band by MS-BLAST HSP hits. Entry
Accession number
Protein name
Organism
Mass (kDa)
Total score
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Q9SQL5 P93801 Q6DTU3 Q71S31 Q9AR78 Q7M1R5 Q9AXH2 O65768 Q6A199 Q8S3V1 O22374 Q9SAT7 Q9ZNQ4 P09678 Q5ZF66 P93258
Superoxide dismutase [Cu–Zn] Superoxide dismutase 4A Superoxide dismutase Copper/zinc superoxide dismutase Putative cytosolic CuZn-superoxide dismutase Superoxide dismutase [Cu–Zn] Cu–Zn superoxide dismutase Superoxide dismutase [Cu–Zn] Cu/Zn superoxide dismutase Copper/zinc superoxide dismutase Cu/Zn superoxide dismutase Copper/zinc-superoxide dismutase Superoxide dismutase Superoxide dismutase [Cu–Zn] Superoxide dismutase [Cu–Zn] Superoxide dismutase [Cu–Zn]
Ananas comosus Zea mays Malus xiaojinensis Citrus limon Populus tremula Glycine max Avicennia marina Carica papaya Helianthus annuus Sandersonia aurantiaca Raphanus sativus Populus tremuloides Cicer arietinum Brassica oleracea var. capitata Plantago major Mesembryanthemum crystallinum
15 176 15 089 15 106 15 099 15 196 15 194 15 281 15 213 15 417 15 273 15 096 15 315 15 222 15 172 15 193 15 173
294 288 286 280 279 277 273 273 271 271 270 270 269 265 264 264
To further support that this UB-DEAE protein from fraction IE-1 is an SOD homologue, SOD activity was evaluated using the NBT assay, a standard test method, in the protein fractions. The result showed that the crude protein precipitate and the IE-1 fraction could both reduce the formation of formazan, presumably by capture of the superoxide radical, and thus exhibit SOD-like activity, with a specific activity of 19.3 and 127.7 U/mg for the crude extract and IE-1 fraction, respectively. Taken together, the data is consistent with the notion that the UB-DEAE antioxidant protein found in the rhizomes of C. comosa is a member of the SOD family. Note however, not only that antioxidant activity was found in the other two principal protein fractions (IE-2 and IE-3) not characterised here, but also that the enrichment of SOD-specific activity from the crude extract to the IE-1 fraction was some 6.6-fold, compared to only a 1.55-fold enrichment of general (DPPH sensitive) antioxidant activity. Thus, whether other antioxidant proteins are present seems likely but remains to be evaluated.
Two reports of SOD enzymes being found in members of the Zingeberaceae plant family have recently come to light. The first involves a Cu–Zn dependent SOD in the leaves and rhizomes of C. longa L., which is stable to high temperatures and harsh conditions (Kochhar & Kochhar, 2008). The second was reported from C. zedoaria Roscoe. (Loc et al., 2008). The discovery of SODs in these two plants are consistent with the finding of a SOD-like antioxidant protein in C. comosa rhizomes reported here, given that this plant is in the same genus as well as family. In conclusion, this study presents the likely confirmation of an antioxidant protein from the SOD enzyme family within the rhizomes of C. comosa. Acknowledgements The authors thank the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Programme (Grant No. PHD/0224/2548), the 90th Anniversary of Chulalongkorn University fund for finan-
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cial support of this research and Ratchadapisek Somphot Endowment Fund (AG001B). The Department of Chemistry, the Faculty of Science, and The Institute of Biotechnology and Genetic Engineering Chulalongkorn University, are both acknowledged for support and facilities. We also, thank Dr. Robert Butcher (Publication Counseling Unit, Chulalongkorn University) for his constructive comments in preparing this manuscript.
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