GREE GRASS JELLY A TIOXIDA TS

5 Table 3.Studies of green grass leaves Aspect Research Result Reference Safety Increasing of lymphocyte cell proliferation on human peripheral blood cells in vitro Pandoyo 2000 Reduction of Cytochrome P420 content and increasing of Glutation STransferase content on mice Sprague Dawley Nugrahenny 2003 Bioavailability Low bioavailability of carotenoid on mice Sprague Dawley Wylma 2003 Low bioavailability of βcarotene content on mice Sprague Dawley Jacobus 2003 Bioavailability of chlorophyll affected by vitamin A content on mice Sprague Dawley Hendriyani 2003 Low bioavailability of flavonoid on mice Sprague Dawley Raharjo 2004 Efficacy Increasing of free radical production by macrofage on mice Balbc Handayani 2000 Reduction of allergy reaction on mice Balbc Rachmini 2000 Increasing of human lymphocyte cell Koessitoresmi 2002 Antioxidant potention to increase life index of lymphocyte cell on mice C3H Setiawaty 2003 Reduction of proliferation on leukemia cancer K 562 and servix cancer Hela Ananta 2000 Reduction of proliferation on breast cancer on mice C3H Rochima 2012 Increasing of SOD content and reduction of catalase on mice C3H Chalid 2003 Cancer Tissue Analysis High HE score of liver tissue on mice C3H Widyanto 2010 Reduction of IHK Caspace3 and Increasing of vascularization marker CD31 tumor cell on mice C3H Aryudhani 2011

C. GREE GRASS JELLY

Leaves of green grass plant used to make a kind of gel obtained from the crushed leaves mixed with water as solvent and would be thickened automatically Sunanto 1995. Green grass jelly can be formed at room temperature, has green color because of its chlorophyll, opaque, and irreversible or the gel can not be form again after being destroyed. Gelling component in green grass jelly is a compound of hydrocolloid. Artha 2001 had succeeded to analyze that component and stated that major component of green grass jelly is a gelling 6 polymers with low methoxyl pectin with Dgalacturonic acid as the main chain with β1,4glycosidic bond and galactose as side chains. It can form a gel chemically with the help of divalent cations minerals. The general structure of pectin molecules can be seen in Figure 1. Figure 1. General structure of pectin Shukla 2011 The suitable degree of acidity pH to produce green grass jelly with the optimum texture is about pH of 47 Garnawati 1978 in Ananta 2000. According to Wyanto 2000, the best concentration to produce green grass jelly is 1:15 or 6.67 wv. The main deterioration of green grass jelly is syneresis, the run out of water from green grass jelly. Syneresis can occur due to the termination of the bond on the fibriller or because fibriler, which is originally located rather far apart to each other, form bonds between fibriler to be close so that the liquid squeezed out Setyaningtyas 2000.

D. HYDROCOLLOIDS

Hydrocolloid is a watersoluble polymer capable of forming a colloidal solution and able to thicken or form a gel from its solution. There are several kinds of hydrocolloids used in this research, i.e. alginate, pectin, and carrageenan.

1. Alginate

Alginate is an unbranched binary copolymer group consisting of Dmannuronic acid residues M and binds β 1,4 with Lguluronic G on some compositions that do not have a repetition Draget 2009. Alginate showed characteristics on affinity for polyvalent cations, especially Ca 2+ ions. This character is similar with pectin. This is influenced by the residual αLguluronic on alginate chain. General structure of the alginate molecule can be seen in Figure 2. Figure 2. General structure of alginate Draget 2009 Alginate gel has a thermoirreversible property, which stable to heat. This means that the alginate gel can be heated without being melted. However, the heating process must be conducted at neutral pH in order to avoid degradation of the alginate structure because it is too acidic or too alkaline. This degradation occurs due to the increased rate of polymer breakdown reaction Draget et al. 1988. 7 Alginate gelation phenomena can be divided into two mechanisms, i.e. diffusion setting and internal setting Draget 2009. Diffusion setting is characterized by gelation occurred because cross linking ion Ca 2+ diffuses from the media into the alginate solution Figure 3a. Internal setting characterized by Ca 2+ ion released in a controlled manner from a particular source of calcium into alginate solution Figure 3b. Gelation mechanism used in this study was internal setting by using the internal sources of calcium in the form of CaCO 3 which can be done on a wide range of pH Draget et al. 1991. By using this gelation system, the use of CaCO 3 in the form of powder is very beneficial because it can increase the surface area of the molecule that can reduce the transition time and gel can be formed more quickly and uniformly. Wyanto 2000 reported that formula with the mixture of 3 green grass extract and 1.75 alginate could provide good gel and preferable organoleptically. a b Figure 3. Gelation mechanisms of alginate a Diffusion setting and b Internal setting Draget 2009

2. Pectin

Pectin is a polimer of galacturonic acid with the chain shape of 14αDgalacturonan with methyl ester bind partially. There are also branch of Larabinose and 14βDgalactan inside Glicksmann 1984. Degree of esterification DE is a ratio of esterified galacturonic acid units with total of galacturonic acid in the molecules. Low Methoxyl Pectin LMP is type of pectin with degree of esterification below 50, while High Methoxyl Pectin HMP is type of pectin with degree of esterification above 50. To form a gel, LMP needs calcium ions because its molecule has high reactivity with calcium ions May 2009. Calcium capability to form insoluble complex is associated with free carboxyl group in the pectin chain. The lower degree of esterification leads to the greater reactivity of pectin chain to the calcium ions. The gel conformation follows the eggbox model, the gel structures are formed due to calcium mineral induction in association with monosaccharide started from axial part of α 1,4Oglycosidic bond from six carbon monosaccharide structure that follow the formation of C1 and 1C, especially on the 8 galacturonic and guluronic acid Walter 1991. The picture of eggbox model is presented on Figure 4. Figure 4. Schematic diagram of eggbox model Grant 1973 in Shukla et al. 2011 Artha 2001 reported that only LMP and alginate could be able to interact synergistically to form a gel with hydrocolloid of green grass jelly. Rustanti 2000 stated that gel product contains of 3 green grass extract and 1.75 LMP has high rupture strength, rupture point, and rigidity. Mixture of green grass extract with LMP high or low DE could better prevent the syneresis on the refrigeration temperature. Camus 2000 stated that mixture of 2 green grass extract and 1.75 hydrocolloid mixture of LMP and alginate 1:1 could provide the best formula on the hedonic and texture characteristics.

3. Carrageenan

Carrageenan is a high molecular weight linear polysaccharide comprising repeating galactose units and 3,6anhydrogalactose 3,6 AG, both sulfated and nonsulfated, joined by alternating α1,3 and β1,4 glycosidic links Imeson 2009. The distinct carrageenan structures differ in 3,6anhydrogalactose and ester sulfate content. Variations in these components influence hydration, gel strength and texture, melting and setting temperatures, syneresis and synergism. These differences are controlled and created by seaweed selection, processing, and blending of different extracts. The ester sulfate and 3,6anhydrogalactose content of carrageenan are approximately 25 and 34 respectively for kappa carrageenan and 32 and 30 respectively for iota carrageenan. Lambda carrageenan contains 35 ester sulfate with little or no 3,6 anhydrogalactose content. Even furcellaran, which in the past has been rather misleadingly called ‘Danish agar’, contains 16–20 sulfate content. These high sulfate levels contrast with agaragar which has very low sulfate content, always below 4.5 and typically it is from 1.5– 2.5. Structure of kappa, iota, and lambda carrageenan are shown in Figure 5. Figure 5. Molecul 2009 If kappa requires heating to rather dark in colo provides elastic ge clear in color and water and solution when the hot solu This process is rev cooling. Campo successive steps; c cause aggregation for gelation to proc are all capable of i more hydrophilic commercial carrag carrageenan is cha network of three adjacent spiral ch lambda carrageena this ‘‘crosslinking” 6. a c olecule structure of kappa a, iota b, and lambda c c appa carrageenan put in cold water, it will swell to for ting to 70°C to dissolve Pebrianata 2005. Gel formed by color and has easily cracked texture Fardiaz 1989. On gel, syneresis free, and reversible Pebrianata 2005. Th and soft in texture Fardiaz 1989. Iota carrageenan poss lution of sodium salt. The ability of kappa and iota carrage t solution is allowed to cool, due to groupcontaining 3,6 is reversible, which means gel will melt when heated and po et al. 2009 stated that gelation of carrageenans invo teps; coiltohelix transition upon cooling and depend on ation between helices. The presence of suitable cation, is an to proceed. For kappa carrageenans, the alkali metal ions L le of inducing gelation. Iota types gel best with calcium ion philic and form fewer junction zones than do kappa carrageenans, kappa and iota are gelforming carrageen is characterized only as a thickener agent. Kappa and iota threedimensional double helices, resulting from the ‘‘ ral chains that contain sulphate groups oriented towards t ageenan, the 2sulphate group is oriented towards the intern nking”. Gelation mechanism of kappa and iota carrageenan 9 b a c carrageenans Imeson to form rough spread that ed by kappa carrageenan is . On the other hand, iota The gel formed is more possesses soluble in cold arrageenans gelation occurs 3,6anhidroDgalactose. and form a gel again after s involves two separate and nd on subsequent cation to , is an absolute requirement ons Li + , Na + , K + , Rb + , Cs + um ions. Iota carrageenan is appa carrageenan. Among ageenans, whereas lambda d iota carrageenans form a he ‘‘crosslinking” of the ards their external part. In internal part, thus avoiding eenan is presented in Figure Figure 6. Gelation In a hot cooled, they inter double helices are Gelation occurs be due to the presenc form a rather firm Miller and Whistle Carrageen antihyperlipidemic potential. Sun polysaccharides is factors. Wijesekar structural features, glycosidic branchi antioxidant activit efficiently and don

E. A TIOXIDA TS

Antioxidants are c processes or reactions that food products, antioxidants foods, oxidation attack su intentionally added to a foo According to Ko antioxidants, especially phe oxygen and reacts with ox functions to break down antioxidants, works by elim ions such as copper and iron elation mechanism of kappa and iota carrageenans Be Mille a hot solution, the polymer molecules are in a coiled sta intertwine in doublehelical structures. As the solution es are believed to nest together with the aid of potassi urs because the linear molecules are unable to form conti resence of structural irregularities. The linear helical porti r firm threedimensional, stable gel in the presence of the a histler 1996. rageenan has biological activities of antitumor, idemic, and anticoagulant activities Campo et al. 2009. I un et al. 2009 pointed out that antioxidant activi des is usually not a function of one single factor, but rat esekara et al. 2011 stated that their antioxidant activ atures, such as degree of sulfating, molecular weight, type ranching. Low molecular weight molecules with high sulfat activity Sun et al. 2009. Low molecules may incorpora d donate proton effectively compared to the high ones Ngo TS are compounds in biological systems that protect against s that lead to excessive oxidation Aryudhani 2011. Mea idants are compounds that can protect materials or food pro such as oxidative rancidity. These compounds can be a food product Fardiaz 1996. Koschhar 1993, antioxidants can be classified into 5 phenolic compounds that can stop free radical chain ox ith oxygen to eliminate oxygen in a closed system, 3 se down fatty hydroperoxide into a final product that is y eliminating solvent oxygen, and 5 chelating agent seque nd iron that catalyzes the oxidation of fat. 10 Miller and Whistler 1996 ed state. As the solution is ution is cooled further, the otassium or calcium ions. continuous double helices l portions then associate to f the appropriate cation Be mor, immunomodulatory, 09. It also has antioxidant activity of marine algae ut rather a combination of activity depends on their t, type of major sugar, and sulfate content had the best orporate to the cells more s Ngo et al. 2011. ainst the harmful effects of Meanwhile, in relation to od products, especially fatty can be naturally present or nto 5 types, ie: 1 primary oxidation of fat, 2 trap secondary antioxidants, at is stable, 4 enzymatic sequestrant, chelates metal One type of antiox compounds that have a sin Wanasundara 1992 state antioxidants, free radicals compounds are flavonoid dihydrocalcone, calcone, flavones, flavonols, flavil antioxidant Harbone 1987 as an antioxidant, phenoli protection from oxidation Pratt and Hudson 1990. 0.17 phenol wb solvent acid at 3.85 wb solvent carotenoids, and chlorophyl Beside the phenol antioxidant activity, namely type of carotenoid that has is also a potential compoun components, such as pheno Jacobus 2003 reported leaves as much as 1, 3 carotene in the liver of mi suggests that the content o bioavailability.

F. CHLOROPHYLL