Environ. Eng. Res. 2012 December,17(S1) : S9-S13 Research Paper http://dx.doi.org/10.4491/eer.2012.17.S1.S9 pISSN 1226-1025 eISSN 2005-968X

Sulphur Free Radical Reactivity of Thioproline and Crude Extracts of timoriana (DC.) Merr.

Robert Thangjam1†, Damayanti Devi Maibam2 1Department of Biotechnology, School of Life Sciences, Mizoram University, Aizawl 796004, Mizoram, India 2Department of Life Sciences, Manipur University, Imphal 795003, Manipur, India

Abstract Parkia timoriana (DC.) Merr. of the family Leguminosae () and sub-family Mimosoideae, commonly known as bean is widely distributed throughout northeast India. It is considered to be a multipurpose tree, with various food and medicinal uses. The seeds are consumed at all developmental stages, and are considered to be a special delicacy due to their distinctive aroma and flavour. The characteristic sulphur aroma of the seeds is associated with the thiol amino acid derivative thioproline, which is a condensation product of formaldehyde and cysteine. In the present study, the presence of formaldehyde and thiol in the different developmental stages of the fruits were evaluated. The level of formaldehyde and thiol content was found to increase with the maturity of the seeds. The generation of sulfhydryl free radicals by thioproline and crude P. timoriana seed extracts on gamma irradiation were observed with the visible destruction of curcumin. The protection of DNA against gamma irradiation in the presence of thioproline in the present study also provides a new lead for investigations into protection against radiotherapy and reduction of risk for exposed individuals.

Keywords: Parkia timoriana, Radioprotection, Suphur free radical, Thioproline

1. Introduction cellular sulfhydryl antioxidant and free radical scavenger, pro- tecting cellular membranes from damage due to oxygen derived Thiol (RSH) compounds are regarded as antioxidant agents as reactions [10-12]. The endogenous formation of thioproline has they protect protein-SH groups against oxidants and can scav- been considered as a detoxification pathway of formaldehyde enge oxygen radicals and other reactive species such as hypo- [13]. Its protection of the liver against various toxic agents, clini- chlorous acid and peroxynitrous acid, thereby preventing harm- cal anti-tumour effects in cancer patients, and anti-aging in dro- ful effects on DNA, intracellular proteins and membrane lipids. sophila has been widely reported [14-16]. Several studies also Thiolic antioxidants such as glutathione (GSH), N-acetylcysteine showed that thioproline administered in the diet stimulates the (NAC) and thioproline are thiol group donors [1, 2]. The deficit in lymphocyte and natural killer (NK) functions in old mice as well these thiolic antioxidants or other thiol group donors can lead to as the macrophage functions in vitro [17]. Furthermore, Thiop- increased lipid peroxidation reactions, with concomitant chang- roline is an effective nitrite-trapping agent in the human body, es in membrane permeability and cellular injury [3]. The dietary thereby inhibiting the carcinogenic N-nitroso compounds. Thio- administration of thiolic antioxidants can prevent an excessive proline has been found in various cooked foods such as cod fish, oxidation of the sulphur pool, especially the progressive oxida- shiitake mushroom and various kinds of cooked vegetables [18]. tion of glutathione and other thiolic compounds with age [4, 5]. In our opinion, these findings justify the attempt to modulate the However, thiols can, themselves, generate free radicals [6]. Sulf- reactivity of thiyl free radicals generated from thioproline on cur- hydryl (thiyl) free radicals can be generated by the irradiation of cumin, and study its effect on the irradiation of DNA. glutathione [7].. Curcumin readily reacts with thiyl free radicals by losing its characteristic visible spectrum and hence providing a reference indicator of sulphur free radical reactivity [8]. 2. Materials and Methods

Thioproline (C4H7NO2S, thiazolidine-4-carboxylic acid), the principal agent responsible for the distinctive sulphur-aroma in 2.1. Estimation of Formaldehyde and Thiol Content Parkia timoriana DC. (Merr.) seeds, is a cyclic sulphur containing amino acid [9]. It is the condensation product of formaldehyde Fresh pod samples of P. timoriana were collected at differ- and cysteine and is a natural metabolite which can act as intra- ent maturity stages (tender, mature, and fully mature). They

This is an Open Access article distributed under the terms of the Creative Received September 09, 2012 Accepted September 29, 2012 Commons Attribution Non-Commercial License (http://creativecommons. † org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, Corresponding Author distribution, and reproduction in any medium, provided the original work is E-mail: [email protected] properly cited. Tel: +91-389-2330859/861 Fax: +91-389-2330644/834

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were rinsed in distilled water and blot dried with tissue paper. proline + 5 mM ascorbic acid, 3) 20 µM curcumin + seed water The outer green skin was scraped and the pulp was used for extract, and 4) 20 µM curcumin + 60% methanol extract, were homogenisation. One gram of each sample (pulp and seeds) of irradiated, respectively at 50, 100, 150, 200, and 250 Gy gamma the uncooked and cooked (boiled in distilled water for 10 min) ray at a dose rate of 0.14 Gy/sec, determined by Fricke dosimetry samples were homogenised in 50 mL distilled water, and were (Instruction manual gamma chamber 4000A; Isotope Group at centrifuged twice at 3,000 rpm for 15 min. The clear superna- Bhabha Atomic Research Centre [BARC], Mumbai, India) using tant was filtered and was immediately used for the estimation a 5100Ci 60Co gamma irradiator (BARC). The absorbances of the of formaldehyde using the method of Nash [19]. 2 mL of the irradiated and non-irradiated solutions were then measured at supernatant was added to 2 mL of reagent containing 2 M am- 420 nm within 5 min of irradiation using 60% methanol as the monium acetate, 0.05 M acetic acid and 0.02 M acetylacetone blank. and was incubated at 37°C for exactly 40 min (99% of the reac- tion was estimated to be completed under these conditions). 2.3. Study of the Effect of Thioproline on Gamma Irra- The absorbance of the yellow colour developed was immediately diation of DNA measured against the sample blank at 412 nm, using a Beckman DU-640 spectrophotometer (Beckman Coulter GmbH, Krefeld, An aqueous solution containing 50 µg/mL calf thymus DNA Germany). The amount of formaldehyde present was estimated and 15 mM thioproline maintained at pH 7.5 was irradiated at using a standard curve of formaldehyde (0.05 µM/mL). Total, 50, 100, 150, 200, and 250 Gy at a dose rate of 0.14 Gy/sec using a protein-bound, and non-protein sulfhydryl groups were assayed 5100Ci 60Co gamma irradiator (BARC). The absorbance was mea- using the method of Sedlak and Lindsay [20]. For the determina- sured at 260 nm for both irradiated and non-irradiated solutions tion of total thiol group, aliquots of 0.5 mL of the homogenates within five min of irradiation with a distilled water preparation were mixed in 15 mL test tubes with 1.5 mL of 0.2 M Tris buffer, acting as a blank. pH 8.2 and 0.1 mL of 0.01 M DTNB. The mixture was brought to All the experiments were conducted with 3 replicates and the 10 mL with 7.9 mL of absolute methanol. The test tube was stop- mean values were calculated. The curcumin depletion factor pered with rubber caps and was allowed to stand with occasional was calculated using the following formula: Curcumin depletion shaking for 30 min, after which it was filtered twice to produce = initial curcumin content (µM) – final curcumin content (µM). a clear filtrate. The absorbance of the clear filtrates was read at Similarly the DNA depletion factor was also calculated with simi- 412 nm in a quartz cell against the sample blank. The amount lar formula of DNA depletion = initial DNA content (µg/mL) – fi- of total thiol present was calculated from a standard curve of L- nal DNA content (µg/mL). cysteine. The non-protein thiol groups were determined by tak- ing aliquots of 5 mL of the homogenates, and mixing with 4 mL distilled water and 1 mL of 5% trichloroacetic acid in 15 mL test 3. Results and Discussion tubes. The tubes were shaken intermittently for 10–15 min and were filtered twice to obtain a clear supernatant. Two milliliter of 3.1. Formaldehyde and Thiol Contents the filtrate added to 4 mL of 0.4 M Tris buffer, pH 8.9 and 0.1 mL of 0.01 M DTNB, and the two were shaken. The absorbance was The formaldehyde and thiol contents at different maturity read against the sample blank at 412 nm within 5 min of the ad- stages of the P. timoriana pods are given in Table 1. Formalde- dition of DTNB by using Beckman DU-640 spectrophotometer. hyde content increases with the maturity stages of the pod with The amount of non-protein thiol was calculated from a standard the maximum content of 0.79 mM/100 g at the fully mature curve of L-cysteine. The amount of protein bound thiol groups kernel. Mature pulp contained higher amount of formaldehyde was calculated by subtracting the non-protein thiol contents than the seed. Cooking resulted in the significant reduction of from the total thiol contents. the formaldehyde content. With advancing maturity stages of the pod, the composition of formaldehyde increased, with the maxi- 2.2. Study of Thioproline for Sulphur Free Radical Reac- mum concentration detected within fully matured seeds. Simi- tivity with Curcumin as Reference Model larly the thiol groups, i.e., total, non-protein and protein bound composition, in P. timoriana increased with the maturity of the The sulphur (thiyl) free radical reactivity was studied by the pods, and decreased upon boiling the preparation. This observa- method of Damayanti et al. [8] and D’Aquino et al. [7]. Thiyl free tion has previously been reported for shiitake mushroom [18]. radicals were generated by gamma irradiation in an air saturated Formaldehyde shows genotoxicity in various in vitro assay sys- solution of thioproline and seed extracts. Prior to this, the ab- tems and the decrease in their content on boiling could be due to sorption spectrum of a solution containing 20 µM curcumin in the volatilisation of formaldehyde and/or the formation of thio- 60% methanolic solution and 15 mM thioproline (pH 4.0) was proline [11, 21]. Endogenous formation of thioproline has been measured at different wavelengths (360, 380, 400, 420, 440, 460, considered as a detoxification pathway of formaldehyde [18]. 480, 500, and 520 nm). Aqueous and methanolic seed extracts The importance of sulfhydryl or thiol groups for the activity of were then prepared by using mature black tree bean seeds cellular enzymes as well as keeping the membrane intact is well soaked in distilled water at room temperature for approximately known [1, 2, 5]. Cellular sulfhydryl groups are the sum of cellular 3 hr, and the outer black coat was removed. Five gram of the seed non-protein (NP-SH) and protein-bound SH (P-SH) groups. Cel- preparation was homogenised in 50 mL distilled water or 60% lular NP-SH groups consist of glutathione, L-cysteine, coenzyme methanol and was centrifuged twice at 7,000 rpm for 10 min. The A, and dipeptides. P-SH groups are found in the biomembranes, supernatant was used for the experiments. For the assessment soluble enzymes and structural proteins. They are important of curcumin destruction by thiyl free radicals 4 different sets of groups having catalytic functions and structural properties. Sulf- experimental solutions were prepared as follows: 1) 20 µM cur- hydryl groups are the sites of radiation damage. Keeping these cumin + 15 mM thioproline, 2) 20 µM curcumin + 15 mM thio- in mind the evaluation of NP-SH and P-SH was conducted. The

http://dx.doi.org/10.4491/eer.2012.17.S1.S9 10 Sulphur free radical reactivity of thioproline and Parkia timoriana

1.2 line and curcumin showed a marked reduction in the depletion level of curcumin. Overall the depletion factors of curcumin in 1.0 the 3 different experimental conditions are in the order of the 0.8 following solutions: curcumin + thioproline > curcumin > cur- 0.6 cumin + thioproline + ascorbic acid. The characteristic chrome orange-yellow color of curcumin is due to the presence of two

Absorbance 0.4 olefinic side chains, which are conjugated to the aromatic ring. 0.2 Its conjugated systems can be destroyed by thiyl free radicals, and in doing so, it loses its characteristic visible spectrum, i.e., 0.0 bleaching. Thus, the destruction of curcumin in the presence of 360 380 400 420 440 460 480 500 520 thioproline, along with its relatively very low destruction in its Wavelength (nm) absence, clearly indicates the generation and reactivity of thiyl free radicals. Similarly, the water and methanolic seed extracts Fig. 1. Absorption spectrum of the solution containing curcumin (20 showed similar patterns of curcumin depletion (Table 3). The de- µM) + thioproline (15 mM). pletion factors of curcumin in the 3 different experimental con- ditions were in the order of the following solutions: curcumin + 60% crude methanolic extract > curcumin + crude water extract decrease may result from the formation of thioproline [9]. > curcumin. It is also interesting to note that the methanolic ex- tract exhibited a far greater degree of curcumin depletion than 3.2. Assessment of Thioproline for Sulphur Free Radical the water extract. The presence of excess organic compounds of Reactivity with Curcumin as a Reference Material known composition, such as methanol and thioproline, in the experimental solutions reduces the chances of organic impuri- Thioproline was assessed for its sulphur free radical reactiv- ties interfering with the formation of thiyl free radicals, which ity through determination of the its absorbance pattern, using react with curcumin. These observations were also reported with curcumin as a reference material (Fig. 1). The maximum absor- the use of methanolic crude turmeric extracts and glutathione, bance was recorded at 420 nm. The levels of curcumin depletion resulting in a visible destruction of the orange-yellow chrome in the solutions containing curcumin, curcumin + thioproline, color of the curcumin [7]. Vitamins C and E can effectively pro- and curcumin + thioproline + ascorbic acid, with different doses tect the destruction of curcumin, which is in agreement with of gamma radiation are given in Table 2. It was observed that the their known reactions with the thiyl free radicals generated from presence of thioproline in the solution contributed to consider- glutathione [7, 8]. This feature of the protection of curcumin able depletion of curcumin upon gamma irradiation, while the from thiyl free radicals in the presence of vitamin C was also ob- addition of ascorbic acid in the solution containing thiopro- served in the present study.

Table 1. Formaldehyde and thiol content at different pod maturity stages of Parkia timoriana Thiol content (mM/100 g) Sample Formaldehyde content (mM/100 g) Total Non-protein Protein Tender pod Uncooked 0.09 ± 0.01 0.37 ± 0.03 0.17 ± 0.01 0.20 ± 0.04 Cooked 0.07 ± 0.01 0.29 ± 0.01 0.11 ± 0.01 0.18 ± 0.02 Mature pod Pulp Uncooked 0.19 ± 0.01 0.36 ± 0.02 0.17 ± 0.01 0.19 ± 0.01 Cooked 0.12 ± 0.01 0.24 ± 0.02 0.13 ± 0.01 0.11 ± 0.01 Seed Uncooked 0.12 ± 0.01 0.41 ± 0.01 0.19 ± 0.01 0.22 ± 0.02 Cooked 0.10 ± 0.01 0.28 ± 0.02 0.16 ± 0.01 0.12 ± 0.01 Fully matured seed Uncooked 0.79 ± 0.06 0.59 ± 0.01 0.37 ± 0.01 0.22 ± 0.03 Cooked 0.43 ± 0.13 0.39 ± 0.01 0.23 ± 0.01 0.16 ± 0.01 Values are presented as mean ± standard error.

Table 2. Effect of gamma irradiation on curcumin in the presence of thioproline and vitamin C Curcumin depletion factor

Radiation dose (Gy) Curcumin only Curcumin (20 µM) + Curcumin (20 µM) + (20 µM) thioproline (15 mM) thioproline (15 mM) + ascorbic acid (5 mM) 0 0.00 ± 0.01 0.00 ± 0.02 0.00 ± 0.03 50 2.85 ± 0.04 3.83 ± 0.05 1.49 ± 0.12 100 3.05 ± 0.01 5.58 ± 0.21 1.68 ± 0.20 150 3.44 ± 0.06 6.94 ± 0.01 1.88 ± 0.01 200 3.83 ± 0.02 9.28 ± 0.04 2.07 ± 0.21 250 4.22 ± 0.11 11.81 ± 0.21 3.44 ± 0.01

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Table 3. Effect gamma irradiation on curcumin in the presence of Table 4. Effect of gamma irradiation on DNA in presence and seed extracts of Parkia timoriana absence of thioproline Curcumin depletion factor Radiation dose DNA depletion factor Radiation Curcumin Curcumin + Curcumin + (Gy) DNA only DNA + thioproline dose (Gy) only water extract 60% MeOH extract 0 0.00 ± 0.01 0.00 ± 0.01 0 0.00 ± 0.02 0.00 ± 0.01 0.00 ± 0.02 50 9.84 ± 0.35 7.47 ± 0.58 50 0.64 ± 0.02 2.23 ± 1.01 6.71 ± 0.40 100 21.85 ± 0.21 8.42 ± 0.22 100 1.12 ± 0.01 2.46 ± 0.04 9.84 ± 1.01 150 23.11 ± 0.11 8.89 ± 0.22 150 2.91 ± 0.11 3.35 ± 0.02 11.40 ± 0.51 200 3.41 ± 0.30 4.24 ± 0.01 11.63 ± 0.21 200 25.95 ± 1.01 9.36 ± 0.33 250 5.81 ± 0.33 6.48 ± 0.01 12.07 ± 0.34 250 29.97 ± 0.01 9.84 ± 0.22

3.3. Effect of Thioproline on Gamma Irradiation of DNA References

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