Journal of the Rubber Research Institute of Sri Lanka (2015) 95, 58-65 Insight into prevention of enzymatic browning and increase of antioxidant potential of crepe rubber with ethephon stimulation A P Attanayake*, L Karunanayake** and A H R L Nilmini* * Rubber Research Institute of Sri Lanka, Ratmalana, Sri Lanka **University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka _______________________________________________________________________ Abstract The effect of different ethephon concentrations 1%-5% (w/v) on colour of crepe rubber and plasticity retention index (PRI) was studied. The study was conducted with the RRISL 121 clone using S/2, d3 harvesting system. The results revealed that ethephon stimulation affects strongly the antioxidant and polyphenol oxidase (PPO) inhibitory potential. Further, thiol content and PRI values were increased while giving low Lovibond colour index upto 3% ethephon concentration and the properties were found to be inferior above 3% concentration. A probable reaction mechanism is proposed for colour improvement with optimum concentration of ethephon. Key words: Ethephon stimulation, Lovibond colour index, plasticity retention index Polyphenol oxidase, Thiol content ____________________________________________________________________ Introduction constituents of latex. Raw natural Latex crepe is the purest form of natural rubber contain about 94% hydrocarbon rubber available in the market and Sri (cis 1- 4, Poly isoprene) and 6% non Lanka is the world’s leading producer rubber constituents such as proteins, of pale crepe and sole crepe for the lipids, carbohydrate (Allen, 1963). It export market (Seneviratne et al., 2003). has been reported that carotenoids, Latex crepe is widely used in rubber tocotrienols, polyphenol oxidase, fatty teats, balloons, condoms, infant items alcohol esters, unsaturated fatty acids, and toys. However, dark colour fatty alcohols, di-glyceride and mono - developed in dry rubber is a major issue glyceride in cause the discoloration in some industries which require a pale natural rubber (NR) (Sakdapipanich, et colour raw material. Therefore, colour is al., 2007). The concentration of one of the most important raw rubber polyphenols, proteins, carotenoids are 2 properties in latex crepe. The colour of X 10-2, 1 and 3 X 10-5 (w/w) the rubber depends on non-rubber respectively and their contribution to the 58 A P Attanayake et al. total absorptivity in the yellow brown the process of fractionation and region are 2.3, 0.1 and 0.01% coagulation, the Frey-Wyssling particles respectively. Out of the total absorbance are disrupted and the enzyme released 90% is attributed to the polyisoprene in into the latex serum. This subsequently rubber molecule (Yusree et al., 2012). leads to discolouration of latex (Schadel The enzymatic discoloration is caused and Walter, 1981). Stimulation by by the naturally occurring phenols and ethephon greatly reduces latex amino phenols in latex (Coupe et al., polyphenol oxidase activity which is 1972). Discoloration due to ethephon known to be correlated positively with stimulation has been reported (Yapa, plugging index, which results in 1976), however there is no evidence for increase of phenolic substances in the effect of ethephon concentration on stimulated latex. Significant increase in colour of crepe rubber. Polyphenol phenolic content reported (after first & oxidases are the key enzymes in the second tapping) after ethephon natural coagulation and darkning of treatment is due to decrease in phenol latex (Brozozowska-Hanower, 1978) oxidase activity (Cretin, 1978). and they are located inside the frey- Brozozowska-Hanower et al., 1978 wyssling particles. Frey-wyssling showed a definite lowering of o- particles are mainly lipid in character diphenol oxidase activity in lattices at and are not present in ammonia the first tapping after stimulation. preserved concentrated latex. Phenolic compounds have been reported Apparently, they are either eliminated to react, after enzymatic oxidation with during centrifuging or they have got thiols such as cysteine (CSH) and dissolved in the serum when the latex is glutathione (GSH) to form colorless ammoniated (d’Auzac, 1989). The product (Mason, 1955). The presence of polyphenol oxidase is responsible for thiols in high concentration in latex act the darkening of coagulated rubber on as an antioxidant hence, it can be used exposure to air or oxygen to improve colour (Yapa, 1976). (Wititsuwannakul et al., 2002). With the Glutathione is an endogenous presence of polyphenol oxidase, phenols antioxidant that plays a major role in and amino phenols combine with reducing reactive oxygen species (ROS) oxygen from air to form orthoquinones. formed during cellular metabolism These orthoquinones react with (https://en.wikipedia.org/wiki/Glutathio naturally occurring amino acids and ne). Therefore a clone with high proteins in latex, giving coloured phenolic content can still yield a compounds resembling melanin colorless or light colour rubber, (Mason, 1955). Both polyphenol provided that it contains sufficient oxidase (PPO) and phenolic substances thiols. Glutathione is known to play a are present in latex but do not react with major role in reducing ROS formed each other intact laticifers. However, in during cellular metabolism. Therefore, 59 New insight into ethephon stimulation the objective of the present study was to replicates. Serum was extracted by investigate the effect of ethephon coagulating 1g of latex with 2.5% tri- concentration on latex discolouration chloro acetic acid (TCA) and volumed and underpinning reasons. up to 25mL. To 2mL of extract, 0.1mL of dithio- bis -nitrobenzoic acid solution Materials and Methods (10 mM) and 2mL tris (0.05M) was Experimental design and location of added and the optical density was the study measured at 412nm within 30min using Six experimental blocks comprising 75 a Jenway 6405 UV/Vis spectrophoto- healthy trees from mono clonal (RRISL meter. The amount of thiol was 121) mature rubber trees were selected estimated against a standard curve from the Galewatta Division, prepared using reduced glutathione. Dartonfield Estate, Agalawatta, Sri Lanka for the experiment. Each block Determination of Lovibond Colour was divided into three replicates Index comprising 25 trees. Trees in each block The test was carried out according to were stimulated with 1.6g of 1%, 2%, ISO 4660 (2011) with three replicates. 3%, 4% and 5% (w/v) ethephon applied Three test pieces were cut from a on the tapping panel using an ¼” paint homogenized UFUB sample using test brush and tapped with half spiral based piece punch and pressed them together once in three days harvesting system by (3.2-3.6 mm thick). The test pieces were keeping one block without stimulation then laminated and pressed in a mould as the control. All the experimental between two sheets of transparent tapping blocks were tapped by the same polyester film, with mould covers tapper in order to avoid tapper superimposed at a pressure of not less variability. than 3.5 MPa for 5min at 150 0C±30C. Freshly tapped latex collected into The 3 test pieces (1.6 mm thick) were vessels, immersed in ice and then send retained in the mould and it was to the laboratory immediately for the compared with standard Lovibond analysis of sucrose and thiol content colour glasses (4/19A) under diffuse without preservation and the other daylight against a matt white fraction was processed into un- background. fractioned un-bleached crepe rubber (UFUB). Determination of plasticity retention index Determination of thiol content in The test was carried out according to natural rubber latex ISO 2930(2009) with three replicates. Analysis of thiol content was carried out The test pellets were cut from a by using colorimetric method developed homogenized sample of UFUB in order by Boyne and Ellman, 1972 with three to get sample thickness of 3.4 mm. The 60 A P Attanayake et al. rapid plasticity number of unaged test performed for basic parameters together pieces and test pieces aged at 140 ± with mean separation by Tukey method. 0.2ºC for 30 min were measured using parallel plate plastimeter. Results and Discussion With s/2 d/3tapping system, the thiol Infra-red Spectroscopy content (antioxidant) and plasticity The rubber samples were extracted with retention index increases up to 3% and acetone for 16 hours and acetone extract above that concentration the properties was sandwiched between two NaCl were drastically reduced with increasing cells and Fourier transform infrared ethephon concentration (Table 1). It is spectroscopy (FTIR) spectra were evident from the results that highest recorded on a Nicolet 380 antioxidant activity showed at 3%, spectrophotometer with 32 scans to hence maximum PRI value also improve signal to noise ratio. obtained at 3% stimulant level. Above 3%, the thiol content decreased. This Data analysis may be due to enhanced in vitro The experimental design was complete synthesis of reactive oxygen species randomized design and data was (ROS). These ROS groups are oxidized analyzed using Minitab 16 statistical glutathione (GSH), leading to decrease software. One-way ANOVA was of their content in latex. Table 1. Variation of Thiol content, Plasticity Retention Index and Lovibond colour Index for raw rubber samples treated with 1%-5% ethephon Ethephon Thiol content Plasticity Retention Lovibond Colour concentration (%) (mmol/g) Index Index 0 0.67e 59.59d 1.5 1 0.78bc 69.06c 1.0 2 0.81b 76.6b 1.0 3 0.88a 83.13a 1.0 4 0.73cd 71.74bc 1.5 5 0.71de 70.43c 1.5 *Means that do not share same letter are significantly different P<0.05 61 New insight into ethephon stimulation According to Lovibond colour index explain that “in a system containing o- 1%-3% ethephon treated samples diphenol and glutathione, the total showed lighter colour than the control oxygen consumption increases sharply sample. 4% and 5% samples are very with increasing thiol content and this bright yellow colour with comparison to sharp break is accompanied by the other samples (Fig. 1). It is evident from marked difference in the colour of the the results that with increasing ethephon final product”.