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RACHMA ALVINIA

  • PSIKOLOGI-13.40.0238.doc
  • 8 MARET.docx
  • 31 MARET.docx

1 RESEARCH METHODOLOGY

  1.1. Materials

  ,distilledwater,gallicacidpowder,

  Materialsforthis research aredriedrosellaflower

  , 1% HCl/MeOH, 2,2-diphenyl-1-picryl hydrazyl (DPPH), 7.5 % sodium

  ethanol carbonate (Na 2 CO 3 ), Folin-Ciocalteus phenol reagent, and solutions pH 4 and 7.

  1.2. Instruments

  , Spectro color meter (Model SE-2000,

  Instruments for this research are Centrifuge

  ku Industries Co., Ltd., Tokyo, Japan), ultrasound shock, pH meter

  Nippon Densho portable , centrifuge tube, Spectrophotometer, beaker glass, micropipette, test tube, scale , paper filter, vacuum filter, vortex mixer, and water-bath.

  1.3. Methods

  1.3.1. Sample Preparation for Rosella Extract The dried rosella was extracted using ratio 1 :3 of solvent (water). A4gdried rosella

  was extracted in 12 0 mL of distilled water in the beaker glass and the samples were (15, 30, 45, and 60 minutes) and three

  heated in waterbath at four different times

  (60, 80 and 100°C). After that, the samples were filtered using

  different temperature filter paper and vacuum filtered. Next , the volume of sample was observed and cooled at room temperature. 1.3.2. pH The pH of the solution was measured by portable pH meter , which had been calibrated with buffer solution pH 4 and 7.

  1.3.3. Color see above ?? CIE (L*, a*, b*) values by a Spectro color meter (Model SE-2000, Nippon Densho ku Industries Co., Ltd., Tokyo, Japan) were calibrated with a white porcelain plate (X=93.06, Y=94.95, Z=112.56). Hue angle was calculated from arctan (b*/a*),

  • , a*, and b* values represent

  with higher values indicating less red color. The L , redness, and yellowness.

  lightness

  1.3.4. DPPH Inhibition Ali quots of the hydrophilic fractions were diluted (1:10) with ethanol, and the assay was

  ,(2007). The sample 0.1 m performed followingthe proceduredescribed by Teowet al. 9 m pipetted into a test tube (three replicates) were mixed using vortex and DPPH 1.

  about 2 0 seconds, and allow stand for 20 minutes in dark condition. The use dark condition because he absorbance of DPPH in methanol and acetone decreases under light ( Sagar & Singh, 2011 ). Absorption at 517 nm was measured (UV-vis

  ) with ethanol as a blank. The procedure was repeated with another

  spectrophotometer

  0.1 m rosella extract with different treatment and distilled water for control. The

  calculation is as follow :

  OD sample

  DPPH Inhibition (%) ¿

  

1 −

×100

  ( OD control )

  1.3.5. Total Polyphenol The test tube containing sample (0.025 m extract rosella and 0.075 m distilled water, three replicates ) added with 1.5 m of Folin-Ciocalteus phenol reagent were mixed and

  k condition. After that, it was added with 1.2 m

  allow to stand for 15 minutes in the dar of sodium carbonate (7.5%), were then mixed, and let stand for 1 hour in the dark

  , the sample was zed (ultrasound shock) about 30

  condition. After 1 hour homogeni seconds. Next , absorption at 765 nm was measured (UV-vis spectrophotometer) with

  k. The procedure was repeated with another 0.025 ml rosella

  distilled water as a blan extract with different treatment and distilled water for control. The total polyphenol content was expressed as gallic acid e quivalents (GAE) in milligrams per gram dry material.

  1.3.6. Total Anthocyanin

  % HCl/MeOH (three

  Prepare centrifuge tube containing 5 ml rosella extract and 5 ml 1 replicates ) were mixed and allow stand for 2 hours in the dark condition. Tubeswere centrifugated (5000 rpm, 15 min, at 4°C), and the clear supernatant was collected. Diluted 1 ml supernatant in 1 ml distilled water and mixed using vortex. Absorption at

  0 nm and 657 nm was measured (UV-vis spectrophotometer) with distilled water as a

  53

  blan k. The procedure was repeated with another 0.025 ml rosella extract with different treatment. The calculation is as follow :

  µ mol T otal Anthocyanin =

  ( g ) (OD 530−0,33 ×OD 657 ) volume extract × dilution × ×100

  31,6 Rosella dry weight

  1.3.7. Data Analysis Data were analy zed by using sing a SPSS package (SPSS 13.0 for windows, SPSS Inc.,

  ) were donebycomparingthemeansbyusing Duncansandat95%confidence

  USA 016. level. The graphs were made by Microsoft Excel 2

2 RESULTS AND DISCUSSION

  (°C)

  2.1. Color and pH Value The color value and pH of rosella extract are shown in Table 1.

  Table 5 -1. The Color and pH value of Rosella Extract Heated at Different Time and Temperature

  (minute)

  • a
  • >b
  • pH

  Time

  9.995±0.304 62.

  15 4 8.941.160 2b 7 0.400±0.000 53.2 95±2.892 2.277 ±0.033 3a 2a 1b 3 45.775 ±3.444 2a 7 0.180±0.156 7 0.180±4.455 2.25 0±0.014 3b 2b 1a

  45 43.6 85±4.292 2a 6 9.740±0.933 63.672.065 2.252 ±0.026 3b 2bc 1a

  6

  45.32

  0±1.230 2a

  6

  090±2.121

  6 54.1 90±0.297 3a 7 0.670±0.255 42.7 80±0.962 2.2 92±0.002 1b 1c 2a

  2.257

  ±0.038 3b 2b 1a

  1

  00

  15 43.5 05±1.379 1b 6 9.405±0.219 5 8.875±7.262 2.25 8±0.026 2a 2a 1b

  3 43. 000±2.079 1a 6 9.030±0.523 63. 885±2.411 2.263 ±0.038 2b 2b 1a

  45 43.3 80±0.537 1a 6 8.575±0.276 63.2 95±1.577 2.252 ±0.002 2b 2bc 1a 6 43.365 ±2.864 1a 6 8.665±0.106 5 9.705±1.605 2.23 8±0.016 2b 2c 1a

  80

  ±0.045 1b 1bc 2a

  Temperature

  ±0.012 1a 1a 2b

  L

  6

  15 71.

  970±0.184 3b 54.

  010±0.283 11.

  822.475

  2.372

  3 5 8.460±0.820 3a 6 8.600±0.325 33.5 95±1.676 2.27 8±0.026 1b 1b 2a

  2.272

  45

  53.74

  0±2.121 3a

  7

  0.910±0.594

  0±3.125

  45.67

  • Numbers are presented as mean ± standard deviation.
  • A number followed by the different of superscript : there was a significant difference at 95% (p<0.05) confidence level based on the univariate test.

  Based on Table 5-1., the L* value of rosella extract heated at 15 minute in all temperature are significantly different. Discoloration wasnt visible on the lightness of the rosella extract. Its effect of temperature extraction was degrade of anthocyanin (pigment of rosella),

  degradation ma ke theanthocyanin becomecolorlessand darker(Hayati,2012). The a * value was suggesting the redness. The a* value of

  rosella extract heated at 15 minute in all temperature are significantly different. The increased of a * value because rosella have red pigment ,(2012),reported rosella

and that pigment will come out if it was extracted. Hayati containednatural constituentsof organic acidssuchas

,citric and3 -indolylaceticacids whichplayedanimportant roleingiving theredcolorof sample extract.

  malic

   Considering the effect of , it was found that the longer extraction time and the higher temperature resulted in the more decrease in a* heated time and temperature value. Chumsri (2008)reported,thatgreaterextractiontemperatureandtimecontributedtolessbrilliantredincolor. The b * value was suggesting the yellowness. The b * value of rosella extract heated at 15 minute in 6C are significantly different.

  The pH of rosella extract was in the range of 2.23 to 2.3

8. The results also showed that rosella extract had lower pH-value. Ithappened (Wongetal.,2002).

  0°C

  becauseofitshighconcentrationoforganicacids The pH value of rosella extract heated at 15 minute in 6 are significantly different. The pH depends on the concentration of free H ionsor mirrored the changes in total organic acids. The free state of H ions is due to dissociation of H ions from the carboxylic group (- COOH) of an organic acid. This increase in pH throughout maturation

  was due to a metabolic process in the fruits that resulted in the decrease of organic acids (Azza et al., 2011). Chumsri et al. (2008) reported,

  that rosellaextracthave alower pH. The result shows that long -time heated and higher temperature will make a pH decreased. Thelower pH valuealsocontributestothe stabilityofred colorinrosella extract , andtheanthocyaninwill stableinlower pHvalue(Hayati,2012). Result also shows that the higher pH will ma ke the L* value higher and it means discoloration of rosella extract more visible than other.

  (2008) reported,thatpHbelow2,theanthocyanin existsprimarilyintheformoftheredflavyliumcation. AsthepHis Chumsri etal.

  raised (>4.5), a rapid proton loss occurred to yield blue quinonoidal, forms at figure 4.

  2.2. DPPH Inhibition -2. The DPPH inhibition of rosella extract is shown in Table 5

  • 2. (%) of Rosella Extract Heated at Different Time and Temperature

  Table 5 DPPH Inhibition Time (minute) Temperature C)

  6 1a 2a 2a

  80

  1

  

00

  15 44.471 ±3.794 87.012.516 89.012±0.965 1b 2b 2b 3 66.565 ±2.070 87.242.204 90.037±1.006 1c 2c 2c

  45 76.5 82±4.119 88.725±1.445 89.860±0.532 1c 2c 2c

  6 76. 013±2.872 89.511.752 89.745±0.151

  • Numbers are presented as mean ± standard deviation.
  • A number followed by the different of superscript : there was a significant difference at 95% (p<0.05) confidence level based on the univariate test.

  Based on the Table 5-2., Anthocyanin of rosella extract heated at 15 and 30 minute in 6C are significantly different. Rosella extract has

  high antioxidant activity. Itsbecauserosellaextract containsantioxidantsaswellasanaturalfoodcolorant(Chumsri etal,2008). The result shows that the higher temperature and longer time will increase DPPH inhibition (%). But 100°C at 60 minutes the DPPH inhibition

  (%) decreased, because the anthocyanin was not stable at a higher temperature and longer heating time degraded anthocyanin. Azza et al., (2011) reported, the polyphenol also contributes to making the higher result of the antioxidant activity (Table of polyphenol at Table 5-3).

  The increase of % DPPH cause by anthocyanin which is antioxidant of rosella and the polarity of antioxidant compounds to solvent due the

  (Azza , 011;Chumsri etal,2008). Thehigh antioxidantactivity observedintherosella couldcausebythe highascorbic extraction et al.

  2 acid contentof thisrosella , polyphenoland otherwater solubleantioxidant (Christian& Jackson,2009;Azza et al. , 2 011). Ivanova et al. , (2005) reported, that Bulgarian medicinal plants have a good correspond between antioxidant activity and phenolic compounds.

  The result show that % DPPH stable at 100 °C along the extraction time. Thishappenscausedtheacidicconditionduringtheextraction, made these wea k chemical associations can augment antioxidant properties increase and anthocyanin stability (Azza et al. , 2 011). The phenolic compounds also can augment anthocyanin stability , depending on temperature different phenolic compound contribute (Kopjar et al. , 2009).

  2.3. Total Polyphenol The total Polyphenol content of Rosella Extract is shown in Table 5 -3.

  Table 5 -3.Total Polyphenol of Rosella Extract Heated at Different Time and Temperature C)

  (minute) (°C)

  Time Temperature

  80

  00

  6 1a 2a 3a

  1 15 -0.002±0.011 0.031±0.026 0.097±0.016 1ab 2ab 3ab 3 0.006±0.005 0.055±0.022 0.094±0.021 1b 2b 3b

  0.012±0.006 0.078±0.003 0.091±0.001

  45 1b 2b 3b

  0.012±0.005 0.080±0.004 0.101±0.008

  6 * Numbers are presented as mean ± standard deviation.

  

:therewasasignificantdifferenceat95%(p<0.05) * A numberfollowedbythe differentofsuperscript

confidence level based on the univariate test. kedelphinidin-3-

  The phenolic content in the plant consists mainly of anthocyanins li

  glucoside , sambubioside, and cyanidin- 3- sambubioside mainly contributing to their

  antioxidant properties (Aurelio et al . , 2007). Tsai & Huang, (2004) reported, that in addition , other phenolic compoundsfound in rosella extract suchas catechin could also react with anthocyanins resulting in complex formation which led to color changes. The result shows that higher temperature and longer time will have higher total polyphenol content. They are all increase along the extraction time cause by the increase of temperature. Sari (2012) reported, that the increase of temperature and total polyphenol have a linear relationship.

  2.4. Total Anthocyanin Total Anthocyanin content of Rosella Extract is shown in Table 5 -4.

  • 4.Total Anthocyanin of Rosella Extract Heated at Different Time and

  Table 5 Temperature idem (minute) (°C)

  Time Temperature

  6 2b 1b 1b

  80

  1

  00

  15 5 8.631±3.056 174. 887±6.625 223.65 8±9.896 2a 1a 1a

  97±13.542 9.471±42.914 08.077±7.113

  3 124.2 2a 1a 1a

  21

  2

  0.846±17.952 0.552±36.370 04.180±19.020

  45

  16 2a 1a 1a

  24

  2 6 155.452.705 226.7227.777 1 94.175±24.961 * Numbers are presented as mean ± standard deviation.

  

:therewasasignificantdifferenceat95%(p<0.05) * A numberfollowedbythe differentofsuperscript

confidence level based on the univariate test.

  Based on the Table 5-4., rosella extract has high Anthocyanin content. Rosellaflower : delphinidin-3-xylosylglucoside and cyanidin-3-

  contains mainly two anthocyanins

  (Aurelio et al., 2007). The result shows that heating time and

  xylosylglucoside temperature made the anthocyanin unstable , and it made the anthocyanin content decreased in the higher temperature or at the longer time. Kahtanet al. (2015) reported that the stability of anthocyanin is correlated with structural features of anthocyanin and

  ,pH,light,andthepresenceofenzymes,phenolic

  alsoaffectedbyfactorssuchasheat

  ,oxygen, sugar, sulfur dioxideandametal ion. The type of anthocyanin can be

  acids seen at Figure 6.

  Heating at 15 6C have a lower yield. Mastuti et al. , (2013) and Suzery et al. , (2010) reported , the lower anthocyanin cause by less thermal exposure time. Long or short

  , contact of solvent and the substance is effect to the yields. The heating condition

  time

  was impactful to the stability of the anthocyanin. Table 5 -4 show that at 6C and 80°C

  there are increase then decrease , while at 100°C only decrease. The increase of anthocyanin at 6C and 80°C happened cause increase of heating temperature have advantage to the extraction because the high temperature will increase the diffusion rate. The solutes solubility of solvent will rise as the increase of temperature , make the extraction rates and yields higher. The longer thermal exposure time also ma ke the diffusion rate increase (Mastuti et al. , 2 013). But at the same time, anthocyanin at 6C

  80°C which a longer thermal exposure time was decrease and at 100 °C all result

  and

  , over exposure time and

  show that anthocyanin was decrease. That happened because high temperature ma ke anthocyanin was degraded. Shaheer et al. , (2014) and Suzery et al. , (2010) reported, anthocyanin degradation happen under heating conditions, accelerated with chalcon formatting longer time of anthocyanin exposure to high

  , furcation of covalent bonds

  temperatures. Degradation is primarily caused by oxidation or increase of oxidation reactions due to thermal processing.

3 CONCLUSION

  Time and temperature of heating contribute to rosella extract content. The longer time and higher temperature made color , pH, total anthocyanin, total polyphenol decrease.

  But the higher temperature and longer time made DPPH inhibition increase. The rosella 80°C for 60 min. That

  extract has acid pH and red color. The best treatment is heating at treatment had a high antioxidant , anthocyanin and red color of the extract.