ARCHIVES

OF BIOCHEMISTRY

Vol. 292, No. 2, February

P,ND BIOPHYSICS

1, pp, 617-623, 1992

Turmerin: A Water Soluble Antioxidant from Turmeric [Curcuma longa] Leela Srinivas,*,l

V. EC.Shalini,*

Peptide

and M. Shylajat

*Department of Nutrition and Food Safety, Central Food Technological Research Institute, MysoreIndia; and TDepartment of Biological Chemistry and Medicine, Jo&n Research Laboratory, 1 Joslin Place, Harvard Medical School, Boston, Massachusetts 02215

013, Karnutaka

State,

Received June 25, 1991, and in revised form September 23,1991

Dietary spice components have been screened for their protective effect against reactive oxygen species (ROS)induced, lipid peroxide-mediated membrane and DNA damage and mutagenecity. A new, water soluble, 5-kDa peptide-Turmerin-from turmeric (Curcuma longa) has been found to be an efficient antioxidant/DNA-protectantlantimutagen. Turmerin forms 0.1% of the dry weight of turmeric and is obtained in a crystalline form. It is a heat stable, noncyclic peptide containing 40 amino acid residues, with a blocked N-terminal and leucine at the C-terminal. It is insensitive to trypsin and pepsin, heat, and uv radiation. Turmerin contains three residues of methionine which are partly responsible for the antioxidant activity. Turmerin at 183 nM offers 80% protection to membranes and DNA against oxidative injury. ROS-induced arachidonate release and the mutagenic activity of t-butyl hydroperoxide are substantially inhibited by Turmerin. Turmerin is noncytotoxic up to milligram concentrations, as tested by Ames assay and o 1992 Academic press, IW. in human lymphocytes.

Biological oxidation, when tilted toward prooxidant states by reactive oxygen species (ROS)2 generators, induces epigenetic and genetic changes, leading to structural and functional losses (l-4.). The ROS-induced lesions are increasingly being recognized as possible causative factors in many diseases (1, 4). Hence it is of paramount impori To whom correspondence should be addressed. ’ Abbreviations used: ROS, re,active oxygen species; SOD, superoxide dismutase; DABITC, 4-N,N’-dimethylaminoazobenzene-4’-isothiocyanate; PITC, phenyl isothiocyanate; Dec.T, decolorized turmeric; PBS, phosphate-buffered saline; BHA, 3-tert-butyl-4-hydroxyanisole; TBS, Tris-buffered saline; TBARS, tlniobarbituric acid reactive substances; FSC, fuel smoke condensate; FA.DU, fluorescence analysis of DNA unwinding; DMSO, dimethyl sulfoxide; TPA, 12-o-tetradecanoyl phorbol 13-acetate. 0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

tance to search for exogenous preventive agents in the form of antioxidants to help, sustain, and spare endogenous antioxidants such as cY-tocopherol, /?-carotene, superoxide dismutase (SOD), and catalase ($6). It has been reported that turmeric and its lipophilic principle curcumin are good antioxidants (7,8). In this communication, we present evidence for the presence of a new, water soluble peptide from turmeric as an efficient antioxidant, noncytotoxic antimutagen, and protectant of DNA, thus providing evidence for the presence of chemopreventive agents in diet, indicating that dietary modulation of prooxidant state-mediated diseases is a real possibility (6). MATERIALS

AND

METHODS

Materials Sephadex G-10 was purchased from Pharmacia United, Sweden. 4N,N’-Dimethylaminoazobenzene-4’-isothiocyanate (DABITC), phenyl isothiocyanate (PITC), t-butyl hydroperoxide, carboxypeptidase B, trypsin, pepsin, pronase, fluorescamine, cyanogen bromide (CNBr), and 12-o-tetradecanoyl phorbol13-acetate (TPA) were purchased from Sigma Chemical Co. Salmonella typhimurium TA 102 tester strain was a gift from Dr. Bruce N. Ames, University of California, Berkeley. Turmeric (Curcuma longa) tubers were purchased from the local market and finely powdered. Dry twigs and dry leaves were collected from nearby places in the months of September-December, stored in polythene bags, and used in a 1:l (w/w) ratio. The moisture content was ~10%. All other reagents and chemicals were of AnalaR grade and were purchased from Glaxo Laboratories, India. Solvents were distilled prior to use. All spectrophotometric measurements were done using a Beckman DU-7B spectrophotometer. Amino acid analysis was conducted using a DuPont amino acid analyzer.

Methods Isolation

of Turmerin

from Aqueous Turmeric

Aqueous turmeric was prepared as explained previously (8). Three grams of turmeric powder was dissolved in 300 ml of boiling distilled water, mixed, vortexed, and centrifuged (1500 rpm, 15 min, 25°C). The clear supernatant (aqueous turmeric) was decolorized on a charcoal col617

618

SRINIVAS,

SHALINI,

umn. Activated charcoal powder was washed thoroughly with 95% ethanol followed by 50% ethanol and distilled water, and packed into a column (100 X 20 mm). Aqueous turmeric (300 ml) was loaded and eluted with two bed volumes of distilled water, followed by 5, 15, and 50% ethanol, the respective fractions were collected, ethanol was evaporated under nitrogen, and the dried contents were dissolved in distilled water and tested for antioxidant activity. The aqueous neutral decolorized fraction which eluted initially had the antioxidant activity. This was denoted decolorized turmeric (Dec.T) and was lyophilized and solubilized in phosphate-buffered saline (PBS, 20 mM, 150 mM NaCl, pH 7.4) to a concentration of 6 mg Dec.T/ml. It was then fractionated on a Sephadex G-10 column as follows: Dec.T (50 mg/6 ml PBS) was loaded onto the column (50 X 3 cm, V, = 100 ml, V, = 300 ml), and eluted with distilled water at a flow rate of 12 ml/h. One-milliliter fractions were collected and monitored at 215 nm, the absorption maxima of peptides (9). Fractions obtained were pooled, lyophilized, solubilized in distilled water, and tested for antioxidant activity. The active antioxidant peptide, Turmerin, was chromatographed on a Dowex 50 H+ column, followed by rechromatography on a Sephadex G-10 column under similar conditions, pooled, lyophilized, and solubilized in distilled water to a final concentration of 20 fig/ml, and scanned for uv absorption between 200 and 300 nm.

Biochemical

Characterization

of Turmerin

Turmerin was tested for glycoconjugates using phenol-sulfuric acid according to the procedure of DuBois et al. (10). The peptide nature was determined by hydrolyzing Turmerin with 6 N HCl, neutralization, and testing with ninhydrin and fluorescamine (11). It was also tested for the presence of -S - and -S-S - groups using nitroprusside by the McCarthy-Sullivan method (12), and for the presence of methionine and cysteine/cystine using CNBr and Ellman’s tests, respectively (13,14). It was also subjected to amino acid analysis (14). The N-terminal amino acid determination was done using the DABITC-PITC coupling method according to the procedure of Chang et a!.. (15) and the C-terminal amino acid analysis was done using carboxypeptidase B followed by amino acid analysis (14). Turmerin was tested for its susceptibility to proteases using trypsin, pepsin, and pronase (14). The peptide was also tested for the presence of fatty acid derivatives, by Folch lipid extraction, and analysis by thin layer chromatography (16). The stability of Turmerin was tested against heat treatment at 100°C for up to 3 h, against storage at 4°C and at room temperature for over a period of 7 days, and against uv radiation at 345 nm for 1 h at room temperature. It was also tested for its solubility in water and organic solvents such as isopropanol, dioxan, acetone, and diethyl ether. The purity of Turmerin was determined by reverse phase high performance liquid chromatography (HPLC), using an acetonitrile:water (70:30 v/v) solvent system with a linear gradient. The peptide (50 rg/lOO ~1 HPLC-grade water with 1% trifluoroacetic acid) was injected into a PBondapak Cis column and eluted with the above solvent system, at a flow rate of 0.5 ml/min, and the fractions were monitored at 215 nm.

Biological

Antioxidant

Activity

of Turmerin

Activity

Turmerin was tested as an antioxidant using the model systems of phosphatidyl choline vesicles, liposomes, and human erythrocyte ghosts, in comparison with BHA and curcumin. The effect of cleaving methionine residues by CNBr treatment (14) and preoxidation (17) on the antioxidant activity of Turmerin was also studied. (i) Phosphatidyl choline vesicles. Vesicles containing 10 Gmol of La-phosphatidylcholine suspended in 1 ml of Tris-buffered saline (TBS, 10 mM, 150 mM NaCl, pH 7.4) were subjected to lipid peroxidation with 10 prnol of ferrous sulfate and 100 Fmol of ascorbic acid (17), at 37’C for 60 min, in the presence and absence of the antioxidants BHA, curcumin at 400 pM (7, 8), and Turmerin at 183 nM. The extent of per-

AND

SHYLAJA

oxidation was monitored by measurement of thiobarbituric acid reactive substances (TBARS), by the method of Stoffel and Ahrens (18). The final concentration of Turmerin was based on dose-response experiments. (ii) Liposomes. Large unilamellar liposomes were prepared using equimolar concentrations of L-cy-phosphatidylcholine and cholesterol, according to the procedure of Szoka and Papahadjopoulos (19). Liposomes of 5 wmol total lipid suspended in 350 ~1 TBS were subjected to peroxidation using the ferrous sulfate-ascorbate system, in the presence and absence of the antioxidants BHA, curcumin, and Turmerin, as explained above. (iii) Human erythrocyte ghosts. Erythrocyte ghosts were prepared from human blood by hypotonic lysis according to the procedure of Dodge et al. (20). The ghost pellets were washed thoroughly with isotonic saline (0.9% NaCl) to remove SOD and suspended in isotonic saline to a protein concentration of 1 mg/ml. Aliquots of ghost suspension corresponding to 300 pg protein (7) were subjected to ferrous sulfate-ascorbate-induced peroxidation for 20 min, in the presence and absence of the antioxidants BHA, curcumin, and Turmerin, as explained above.

DNA-Protectant

Activity

Turmerin was studied for its DNA-protective activity against the oxidative damage induced by uv radiation and lipid peroxides, using calf thymus DNA. Household fuel smoke condensate (FSC), prepared by smouldering twigs and dry leaves, was used as yet another source of ROS against human lymphocyte DNA. (i) Ultraviolet radiation. Calf thymus DNA (1 mg/ml PBS, pH 7.4) was subjected to uv radiation (345 nm, Hanovia lamp) for 60 min at 25”C, in the presence and absence of the antioxidants BHA and curcumin at 400 pM and Turmerin at 183 nM, in 1 ml PBS. At regular time intervals, 200-pl aliquots were drawn, and added to 3 ml of ethidium bromide solution (0.5 wg/ml trisodium phosphate buffer, 20 mM, 100 pM EDTA, pH 11.8) and mixed well and the fluorescence of the solutions was measured at 520 nm excitation and 590 nm emission (21). Appropriate blanks and controls were included to rule out nonspecific interactions. (ii) Lipid peroxides. Lipid peroxide-induced DNA damage was studied by the measurement of a soluble, DNA damage-specific, sensitive fluorescent product (Em, 425 nm), according to the method of Fujimoto et al. (8, 22). The DNA-protective activity of Turmerin (183 nM) was studied in comparison with BHA and curcumin (400 PM). (iii) Fuel smoke condensate. Turmerin was also tested as a DNAprotectant in comparison with BHA and curcumin, against FSC-induced oxidative damage in human lymphocyte DNA, as studied by fluorescence analysis of DNA unwinding (FADU) (23). Turmerin was used at 183 nM while BHA and curcumin were used at 40 gM final concentrations, against FSC at 1:lOO dilution (24, 25).

Antimutagenic

Activity

of Turmerin

The antimutagenic activity of Turmerin was studied by the Ames assay, using S. typhimurium TA 102 tester strains which detect oxidative mutagens. The TA 102 strain contains the ochre mutation in the hisG gene. This strain efficiently detects a variety of mutagens such as formaldehyde, glyoxal, various hydroperoxides, bleomycin, phenylhydrazine, X rays, uv light, streptonigrin, and crosslinking agents such as psolarens and mitomycin C. The strain is characterized by another mutationrfa-which causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules such as benzo[a]pyrene that do not penetrate the normal cell wall. The other mutation-uvrB-is a deletion of a gene coding for the DNA excision repair system, resulting in greatly increased sensitivity in detecting many mutagens (26, 27). The mutagenecity in this strain was induced by a lipid peroxide, viz., t-butylhydroperoxide (100 fi~/lOa cells/plate). Turmerin was used at 183 nM and BHA was used at 400 PM. The endpoint of the assay was the formation of histidine-revertant

TURMERIN:

ANTIOXIDANT

PEPTIDE

FROM

TURMERIC

619

colonies in the presence and absence of the oxidant/protectants. Appropriate controls were included to rule out false positives and negatives.

Turmerin-DNA

Interactions

The possible interaction of Turmerin with DNA was studied to understand its DNA-protective activity, using calf thymus DNA. Calf thymus DNA (450 pg/ml PBS) was sheared using a 20-guage needle and incubated with Turmerin (1.7 nmol) at 37’C for 20 min and the solution was then fractionated on a ISephadex G-10 column (50 X 1 cm) and eluted with PBS (flow rate: 12 ml/h, V, = 15 ml, V, = 45 ml). Onemilliliter fractions were collected and monitored for uv absorption at both 215 nm (peptide) and 260 nm (DNA). Untreated DNA and peptide solutions were also fractionated and monitored under similar conditions.

Turmerin

as an InhibitSor of the Arachidonate

Cascade

The inhibitory effect of Tnrmerin on TPA-induced arachidonate release was studied by the mouse ear assay (28). Swiss mice (female, 8 weeks old, six animals per group) were painted on the ear with 10 ~1 acetone (control) or 600 ng of Turmerin in 10 (~1acetone, 30 min prior to the application of TPA (1 p:g/lO ~1 acetone). The mice were sacrificed 5 h later by cervical dislocatison and the ears were weighed (28).

RESULTS

The active antioxidant component of aqueous turmeric extract was isolated by decolorization and fractionation on a Sephadex G-10 column. Figure 1 shows the elution profile of the decolorized extract of turmeric on the Sephadex G-10 column, indicating two peaks-a minor peak of higher molecular weight designated as FI and a major peak of lower molecular weight designated as FII, as monitored by the uv absorption of the fractions at 215

I----

Cl ”

,

LIFE

so

120

160

200

240

I

280

Elution volume (ml) FIG. 1. Isolation of Turmerin from turmeric. Aqueous turmeric extract (3 mg/ml hot distilled water) was decolorized on a charcoal column (100 X 20 mm) and eluted with 2 bed volumes of distilled water and 5, 10, and 50% ethanol; fractions were collected and ethanolic fractions were evaporated under nitrogen and tested for antioxidant activity. The active antioxidant neutral aqueous fraction-Dec.T-was lyophilized in PBS (20 mM, 150 mM NaCI, pH 7..4) to a concentration of 6 mg Dec.T/ml, fractionated on a Sephadex G-10 column (50 X 3 cm, V, = 100 ml, V, = 300 ml), and eluted with water (12 ml/h), and l-ml fractions were monitored at 215 nm. FI-minor high-molecular-weight fraction, FIImajor low-molecular-weight fraction. The active fraction (FII) was pooled, lyophilized, solubilized in water, and rechromatographed (0). Values are means of four replicates, the range of which was