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Cosmetic Science Research Bulletin Vol. (No.

), xx – yy, 2016

Effect of light, pH and temperature on color and antioxidant activity of


beetroot (Beta vulgaris L.) extract

Atitan Chudpimai and Mayuramas Wilai*


School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, Thailand
*Corresponding author. E-mail: Mayuramas@mfu.ac.th

Abstract

This research aimed to study the effect of light, pH and temperature on the colorant and antioxidant
activities of Beetroot (Beta vulgaris L.) extract. Beetroot were extracted by the maceration technique. The
extract was dried by freeze drying and spray dry method. The colorant of freeze dried and spray dried
extract were performed by Ultrascan. The extracts were stored for 3 weeks at different conditions such as
light, pH and temperature. The best storage condition for keeping stability of beetroot (Beta vulgaris L.)
extract is pH3 and 4°C. The antioxidant activity of extract were performed by 2,2 -diphenyl-1-
picrylhydrazyl radical (DPPH) assays. The IC50 value of ascorbic acid and beetroot extract from freeze
drying and spray drying method before the stability test were 22.03 µg/ml, 290.67 µg/ml and 323.38 µg/ml
respectively. After stablity test, the ability of antioxidant activity of beetroot extract was decreased
individually according to each condition so the condition that beetroot extract’s antioxidant activity
decreased least is the condition without light exposed, 4°c and pH3. Therefore, beetrot extract can be used
as active ingredient and color additive in cosmetic product.

Keywords: Antioxidant activity, Free radical scavenging activity, Beetroot (Beta vulgaris L.)

1.Introduction originally from Europe and North Africa (Gokhale and


Natural colorants are considered as safe substance Lele, 2014). It contains phenolic acids including p-
and they are mire desirable than synthetic ones for coumaric, protocatechuic, ferulic, vanillic, p-
commercial application as additive in cosmetic. Color hydroxybenzoic and syringic acids (Georgiev et al.,
from natural pigment is one of the acceptable factors 2002). It also plays a role in free radical scavenger,
which considered quality and defined product also known as antioxidant , which prevents active
acceptance. People increasingly prefer natural oxygen-induced and free radical-mediated oxidation
pigment to synthetic colorant. In fact, The USA of biological molecules or delay the oxidation
permitted list of synthetic colorants was reduced from processes
700 to only seven until the beginning of the XXI In fact, the interest of beetroot increased because
Century due to their negative effects on the of its color and antioxidant activity (Georgiev et al.
environment and people including allergy and toxic 2002). Beetroot has been cultivated for hundred years
(Azeredo, 2009). Currently, the interest in using fornits juice and extract for traditional medicine and Commented [U1]: Need to find new reference to suppo
natural pigment has increased because of its non- cosmetic additives in the form of beet juice and beet your information ka
toxicity and health benefit, mainly as antioxidant. powder (Chawla, et al., 2015).
However, the disadvantages of natural colorants are Beetroot is a potential source of betalains which Commented [KWV2R1]:
their instability to light, oxygen, pH, temperature and are water soluble nitrogenous pigments that are
water activity. yellow, orange, pink, red and purple colored and
Beetroot (Beta vulgaris L.) is a popular vegetable composed of two main groups, red-violet colored
in many parts of the world. It is classified as a root betacyanins and yellow-orange colored betaxanthins
vegetable from Chenopodiaceae family which is (Azeredo, 2009).

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Figure 1 The structure of betaxanthin, betelain and betacyanin

According to Jackman and Smith (1996), the with tap water and cut into 1cm x 1cm cube pieces. It
reduction with the closed structure of cyclo-Dopa was stored at 4℃ until use.
provides the electronic resonance to the diphenolic
aromatic ring. This extra conjugation shifts the 2.2 Preparation of the plant extract
maximum absorption from 480 nm (yellow,
betaxanthins) to about 540 nm (violet, beacyanins). The 1.6 kg of Beetroot was used to extract by
Betalains replace the anthocyanins in most of the plants maceration technique. The beetroot was soaked in 45%
in the order Caryophyllales (Strack, Vogt and ethanol at a ratio 1:3 (w/v) and incubated in incubator
Schliemann, 2003). Betalain is sensitive to light, pH, shaker at 225 rpm, for 24 hours at 30 ℃ thrice to get
temperature and water activity (Stintzing & Carle, the most extract. The mixture was filtered by suction
2004) so its storage condition in solution form is filtration method and concentrated by a rotary
inappropriate (Azeredo, 2009) (Figure 1). evaporator under vacuum at 50°C (Ravichandran et al.,
Accordingly, the popular methods to remove water 2013).
component from emulsion or suspension for a better
stability while storage are freeze drying and spray 2.3 Chemical Substances
drying. Spray drying is the atomizing of an emulsion or Ascorbic acid (Sigma-Aldrich, USA), Ethanol
suspension in a drying chamber with hot air circulation. (Sigma-Aldrich, USA), Hydrochloric acid 37%
The solution is contacted with hot air which cause the
(Sigma-Aldrich, USA), Sodium chloride (Sigma-
water. Spray drying has good character reconstitution,
Aldrich, USA) were used in this study.
low water activity which is suitable for transport and
storage (Khan et al., 2010). The advantages of spray 2.4 Reagents
drying are low costs and short contact time (Sagar and
Kumar, 2010). Spray drying can generate sticky 2,2-diphenyl-1-picrylhydrazyl (Sigma-Aldrich,
products sometimes (Hennigs et al., 2001). Another USA) were purchased for this study.
technique freeze drying, which forms frozen condition
of the solution followed by drying sublimation under 2.5 Equipments
vacuum, and the solid directly changes into ice in vapor
without becoming liquid (Kumar et al., 2011). The 2 digits’ balance, and 4 digits’ balance (Ohaus,
According to the major composition of the extracts USA), Beaker 50, 100, 250, 500 mL (Durex,
from Beetroot as Betalains, it has been reported to be Germany), Dropper with Rubber (Pyrex, Germany),
antioxidant (Tomasz, 2016). Therefore, this extract Freeze dryer (Labconco, USA), Glass Funnel (Pyrex,
may posses as cosmetics ingredient for antioxidant and Germany), Incubator shakier (Sithiporn Associates
color properties (Kapadia & Rao, 2012) . The purpose Co., Ltd, Thailand), Laborator bottle (Durex,
of this study is to investigate and determine the effect Germany), Micropipette (Thermo Fisher Scientific,
of light, pH and temperature on the color and USA), Micropipette Tips (Thermo Fisher Scientific,
antioxidant activity from Beetroot extract by spray USA), Microplate Reader (BMG, Australia), Rotary
drying and freeze drying for color additive evaporator (Eyela,N-1000 from Sithiporn Associates
development in cosmetic. Co., Ltd, Thailand), Round Bottom Flask 500 mL
(Pyrex, Germany), Spectrophotometer (Sithiporn
2. Materials and Methods Associates Co., Ltd, Thailand), Spray dryer (JCM Best
2.1 Plant collection Technology Co., Ltd., Thailand), Test tube (Pyrex,
The fresh beetroot was bought from convenient Germany), Test tube, Stirring rods (Pyrex ,Germany),
store in Nakhonratchasima, Thailand. It was washed UV-Visible spectrophotometer (Biochrom, England),
Ultrascan VIS (Hunterlab, USA), Volumetric flasks

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(Isolab, Germany), 96-well plate (Thermo Scientific) and 9 covered with aluminium foil to avoid the effect
were used in this study. of light and kept at three different temperatures: 4°C,
25°C and 45°C for 3 weeks. Fume hood, hot air oven,
2.6 Drying methods refrigerator and water baht were used.

2.6.1 Freeze drying 2.9.3 Evaluation of color


The sample before and after being kept in different
Pre-freezing was performed by FreeZone Benchtop
conditions were evaluated using Ultrascan VIS in
Shell Freezer at -30°C. The sample was frozen by terms of Hunter ‘L’ (lightness), ‘a’ (redness and
FreeZone Benchtop Freeze Dryer for 5 days to ensure greenness) and ‘b’ (yellowness and blueness). The
complete drying. The pressure was reduced to 0.47 instrument was calibrated with standard white and
mBar and the temperature was reduced to -40°C or black tiles. All the experiments were done in triplicates.
lower. The final sample was stored in glass bottle and
covered with aluminium foil for later use. 2.9.4 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
assay
2.6.2 Spray drying
According to the method described by Sasa et al.
According the the applied method described by (2012), antioxidant activity of Beetroot extract before
Bazaria et al. (2016), the sample was dried on a JCM and after all different testing conditions were
minilab spray dryer. The drying conditions were: determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
drying air inlet temperature of 200°C and outlet 80°C; method with slightly modification of Straus et al.,
atomization pressure: 0.036 psi; average flow drying 2012. The 0.1mM of DPPH solution was freshly
air of 15 litres/hr. The final sample was stored in plastic prepared in ethanol. 50 µl of the extract was added to
bottle and covered with aluminium foil for later use. 150 µL of 0.1 mM DPPH solution. The mixtures were
mixed and kept under dark condition at room
2.7 Spectrophotometric determination of Phenolic temperature for 30 min. The absorbance (ABS) was
compound in Beetroot Extract measured by spectrophotometer at 520 nm. Ethanol
The stock solution of Beetroot extract was prepared was used as a blank. All the experiments were done in
at concentration of 4 mg/ml in DI water at 4°C and then triplicates. The scavenging ability of the extract and Commented [U3]: I think this unit must be mg/ml rather
diluted into the concentration range of 31.25 to 4,000 ascorbic acid toward DPPH radical was calculated by
than ug/ml ka.
µg/ml. The UV spectrophotometric determination of using Scavenging effect equation %: (A0 – A1 ) x 100%
the extracts was scanned at the wavelength of 200-600 /A0, where A0 is absorbance of the control reaction and
nm. A1 is absorbance in presence of the extract or ascorbic
acid. The half maximal inhibitory concentration (IC50)
was calculated graphically using a linear range of a
2.9 Stability test calibration curve by plotting the extract concentration
The 1 µg/ml sample solution in DI water was kept versus the corresponding scavenging effect.
in different conditions for 3 weeks. The stability of the
samples was measured by the colorant and the 2.10 Statistical analysis
antioxidant activity of the sample before and after The IBM SPSS Statistics 22 program (trial version)
being kept in different conditions. was used for data analysis. The data were analyzed and
compared by t-test. The level of statistical significance
2.9.1 Stability test on light and temperature was taken at p-value of less than 0.05 levels.
According to the method described by Chandran et
al. (2012) and Woo et al. (2011), the effect of light and 3. Results and Discussion
temperature on colorant stability was done with 3 ml of 3.1 Extraction and Drying methods Commented [U4]:
samples solution inside test tube covered with and Beetroot was extracted by maceration, then was
without aluminium foil, sealed with parafilm and kept dried by freeze drying and spray drying method.
at three different temperatures: 4°C, 25°C and 45°C for
Commented [U5]: เอาไปอยูใ
้ น Result and discussion
The percentage yield of the crude extract was 3.057%.
3 weeks. Fume hood, hot air oven, refrigerator and The extract has red color and sticky feeling. The extract
water baht were used. The extract also was exposed for from freeze drying is less sticky than the one from
heat-cold cycle for 6 cycles. (Molina, Hernández- spray drying.
Martínez, Cortez-Valadez, García-Hernández &
Estevez, 2014). 3.2 UV/Vis Spectrophotometry
According to research by Khoo et al. (2011)
2.9.2 Stability test on pH and temperature including the report from Zryd et al. (2003) which
According to the method described by Wong et al. characterized the character of carotenoids,
(2014), the effect of pH and temperature on colorant betaxanthins and betacyanins under UV/Vis
stability was performed with 3 ml of samples solution
spectrophotometer exhibited maximum absorption at
inside test tube prepared in four different pH: 3, 5, 7
λmax between 330 - 350, 470 - 486 and 535 - 550 nm,

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respectively. From Figure 2, there was the UV/Vis 1.50
500 µg/ml λmax = 480 nm
spectra of the beetroot extracts shown the maximum
1000 µg/ml
absorption at λmax at 326, 484, and 541 nm which might 1.00 2000 µg/ml
be able to characterized as carotenoids, betaxanthins 4000 µg/ml

and betacyanins, respectively.

Absorbance (AU)
0.50

4.50 0.00
λmax = 484 nm 200 250 300 350 400 450 500 550 600
4.00
3.50 λmax = 541 nm
-0.50
3.00
Absorbance (AU)

λmax = 326 nm
2.50
2.00 -1.00
Wavelength (nm)
1.50 (A)
1.00
2.50
0.50
0.00 500 µg/ml λmax = 480 nm
2.00
-0.50 200 250 300 350 400 450 500 550 600 650 700 750 800 1000 µg/ml

-1.00 1.50 2000 µg/ml


Wavelength (nm) 4000 µg/ml
Absorbance (AU)

1.00
Figure 2 The UV/Vis spectra of beetroot extract.
0.50

After drying process via freeze dried and spray dried


0.00
methods, the beetroot powders were determined using 200 250 300 350 400 450 500 550 600
UV/Vis spectrophotometer which exhibited maximum -0.50
absorption spectra’s at λmax 480 nm where the
-1.00
absorption peak at λmax 362 and 541were decreased
(Figure 3). It would be estimated that the drying -1.50
Wavelength (nm)
process both freeze dried and spray dried methods (B)
effected the stability of the compounds. Figure 3 UV/Vis spectra of the beetroot extract from
feeze drying (A) and spray drying (B) procedures at
3.3 Stability test on colorant 540 nm.
From table 1, it was observed that both beetroot
extract from freeze drying and spray drying were From table 2, it was observed that there was a
lighter. There was significant change (p<0.05) in the consistent change in Hunter ‘L’ 'a' and 'b' which
hunter ‘L’ of both freeze dried and spray dried extract represent the lightness, redness and yellowness through
after 6 hot-cool cycles but there was a significant the different temperature. With the increase of
decrease of the hunter ‘a’ only from freeze dried extract temperature and time, the hunter ‘L, ‘a’ and ‘b’ of the
which mean the extract from spray drying is more freeze dried and spray dried extract which exposed to
stable in different temperature than the extract from light in 25°C and 45°C significantly changed (p<0.05),
freeze drying. There was no significant change in the the hunter ‘L’ increased and the hunter ‘a’ and ‘b’
hunter ‘b’ of both freeze dried extract and spray dried decreased except the hunter ‘b’ of freeze dried extract
extract. and hunter ‘a’ and ‘b’ of spray dried extract in 4°C did
not decreased significantly (p>0.05).

Table 1 The colorant of freeze dried and spray dried beetroot extract by Ultrascan VIS with hot-cool cycles

Cycle L* ±SD a* ±SD b* ±SD


Freeze dried Spray dried Freeze dried Spray dried Freeze dried Spray dried
0 27.49 ± 0.43** 26.2 ± 0.47** 2.59 ± 0.23** 2.41 ± 0.13 4.56 ± 0.22 4.07 ± 0.29
1 23.05 ± 0.05 21.47 ± 0.38 1.42 ± 0.16 1.71 ± 0.21 3.8 ± 0.19 4.18 ± 0.42
2 21.4 ± 0.15 21.01 ± 0.95 1.64 ± 0.23 1.85 ± 0.09 4.82 ± 0.24 4.48 ± 0.29
3 21.04 ± 0.32 20.25 ± 0.52 1.93 ± 0.14 2.55 ± 0.14 4.33 ± 0.12 4.18 ± 0.06
4 21.42 ± 0.26 21.54 ± 0.36 1.31 ± 0.06 1.71 ± 0.13 4.23 ± 0.06 4.52 ± 0.17
5 21.68 ± 0.06 21.62 ± 0.28 1.16 ± 0.04 1.76 ± 0.17 4.74 ± 0.32 4.44 ± 0.41
6 21.67 ± 0.1** 19.03 ± 0.3** 1.22 ± 0.13** 1.75 ± 0.22 4.57 ± 0.52 3.65 0.31
*L = lightness, -a = greenness, +a = redness, -b = blueness, +b = yellowness
**The colorant was changed significantly (p<0.05) comparing the colorant of the extract before anf after 6 cycles

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Table 2 The colorant of freeze dried and spray dried beetroot extract by Ultrascan VIS with and without light
exposed conditions.

Temp Week L*±SD a*±SD b*±SD


(C°) Light Dark Light Dark Lighty Dark
Freeze 4 0 27.85 ± 0.23** 27.13 ± 0.24** 2.36 ± 0.22** 2.76 ± 0.06** 4.07 ± 0.15 4.45 ± 0.11**
Dried 1 21.97 ± 0.99 25.09 ± 0.55 1.89 ± 0.27 2.02 ± 0.08 3.97 ± 0.29 4.10 ± 0.64
2 25.27 ± 0.61 25.27 ± 0.20 2.53 ± 0.23 2.55 ± 0.26 5.28 ± 0.24 5.27 ± 0.10
3 21.55 ± 0.29** 23.11 ± 0.44** 1.65 ± 0.17** 2.49 ± 0.07** 4.19 ± 0.61 5.57 ± 0.18**
25 0 25.98 ± 0.13** 27.12 ± 0.79 2.76 ± 0.39** 2.66 ± 0.04** 4.66 ± 0.28** 4.32 ± 0.31
1 26.85 ± 0.99 22.93 ± 0.08 0.94 ± 0.18 1.08 ± 0.19 3.26 ± 0.84 4.70 ± 0.67
2 28.24 ± 0.14 28.16 ± 0.42 -0.39 ± 0.04 -0.21 ± 0.11 2.81 ± 0.22 3.43 ± 0.15
3 28.16 ± 0.53** 28.13 ± 0.36 -0.36 ± 0.15** -0.66 ± 0.15** 2.12 ± 0.03** 3.68 ± 0.28
45 0 26.71 ± 0.43** 27.64 ± 0.40 2.54 ± 0.16** 2.44 ± 0.13** 4.68 ± 0.79** 4.40 ± 0.31**
1 24.91 ± 0.63 24.99 ± 0.38 -0.25 ± 0.01 -0.14 ± 0.10 3.49 ± 0.30 3.51 ± 0.12
2 28.79 ± 0.23 27.71 ± 0.43 -0.51 ± 0.08 -0.07 ± 0.14 1.81 ± 0.52 1.80 ± 0.38
3 29.63 ± 0.18** 27.62 ± 0.16 -0.34 ± 0.04** -0.44 ± 0.11** 1.74 ± 0.66** 2.43 ± 0.16**
Spray 4 0 27.85 ± 0.23** 25.57 ± 0.25** 2.18 ± 0.15 2.53 ± 0.20 4.07 ± 0.15 3.25 ± 0.49**
Dried 1 21.59 ± 0.46 24.29 ± 1.79 2.38 ± 0.12 2.12 ± 0.12 3.46 ± 0.16 3.17 ± 0.28
2 26.24 ± 0.45 23.96 ± 0.60 2.64 ± 0.19 2.96 ± 0.31 4.78 ± 0.48 4.18 ± 0.26
3 21.28 ± 0.61** 23.48 ± 0.36** 1.89 ± 0.36 3.11 ± 0.09 3.13 ± 0.54 5.49 ± 0.62**
25 0 25.98 ± 0.13** 26.32 ± 0.82** 2.76 ± 0.39** 2.7 ± 0.09** 4.66 ± 0.28** 4.17 ± 0.53
1 27.53 ± 0.07 25.59 ± 2.27 0.88 ± 0.26 1.63 ± 0.60 3.14 ± 0.75 1.63 ± 0.60
2 28.69 ± 0.33 28.26 ± 0.55 -0.46 ± 0.09 -0.24 ± 0.13 2.48 ± 0.32 -0.24 ± 0.13
3 29.08 ± 0.31** 26.26 ± 0.49 -0.42 ± 0.07** -0.66 ± 0.25** 2.09 ± 0.19** 3.64 ± 0.28
45 0 26.71 ± 0.43** 27.12 ± 0.20** 2.54 ± 0.16** 2.62 ± 0.30** 4.68 ± 0.79** 4.04 ± 0.30
1 24.33 ± 0.64 24.36 ± 0.37 -0.14 ± 0.15 -0.64 ± 0.06 4.29 ± 0.32 4.66 ± 0.40
2 27.09 ± 0.69 28.57 ± 0.43 -0.30 ± 0.12 -0.35 ± 0.07 2.68 ± 0.54 1.92 ± 0.62
3 25.78 ± 0.60** 26.06 ± 0.13** -0.04 ± 0.09** -0.06 ± 0.03** 1.70 ± 0.16** 2.55 ± 0.55
*L = lightness, -a = greenness, +a = redness, -b = blueness, +b = yellowness
**The colorant was changed significantly (p<0.05) comparing the colorant of the extract in week0 and week3

Also, it was observed that the hunter ‘L’ and ‘b’ of spray dried extract in the condition of 25°C, 45°C and
the freeze dried and spray dried extract without light pH7 changed significantly. For pH9, all the hunter of
exposed in 25 °C did not change significantly (p>0.05) both freeze dried and spray dried extract changed
while the same hunter in the same temperature with significantly except the hunter ‘b’ of freeze dried
light exposed decreased significantly (p<0.05). The extract in 4°C, the hunter ‘L’ of spray dried in 4°C and
hunter ‘L’ of the freeze dried extract (p>0.05) and the the hunter ‘L’ of freeze dried in 25°C. Therefore, the
hunter ‘b’ of spray dried extract (p>0.05) in 45°C did conditions which are suitabke for stability of extract’s
not change significantly which mean the light truly colorant are 4°C, pH3 and pH7.
plays an important role in affecting the colorant of the
extract. 3.4 DPPH assay
From table 3, it was observed that the Hunter ‘L’’a’ The determination of DPPH assay, ascorbic acid
and ‘b’ of both freeze dried and spray dried extract in was used as a standard to set a standard curve
the condition of 4°C and pH3 did not change y=2.2118x+1.1547, R2 = 0.999. The IC50 value of
significantly while the Hunter’L and ‘a’ of both extract ascorbic acid and beetroot extract from freeze dry and
in 25°C, 45°C and pH3 changed significantly. There spray dry before the stability test were 22.03 µg/ml,
was significant change in the hunter ‘L’ of both freeze 290.67 µg/ml and 323.38 µg/ml respectively. The
dried and spray dried extract in 4°C, pH5 and the interaction between the antioxidants and DPPH radical
hunter’a’ of both freeze dried and spray dried extract in results the reduction of DPPH radical into the DPPH
the condition of 25°C, 45°C and pH5 changed form which consequence the absorbance’s decrease
significantly. from the DPPH form (Harborne, 1998). The radical to
There was no siginificant change of the hunter ‘L, the DPPHH form results in decolorization (yellow
‘a’ and ‘b’ of both freeze dried and spray dried extract color) with respect to the number of electrons captured.
in 4°C, pH7 but the hunter’a’ of both freeze dried and

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Table 3 The colorant of freeze dried and spray dried beetroot extract by Ultrascan VIS in pH3, 5, 7 and 9
conditions.

Temp. Week L* ±SD a* ±SD b* ±SD


(°C) Freeze dried Spray dried Freeze dried Spray dried Freeze dried Spray dried
pH3 4 0 26.24 ± 0.19 22.38 ± 0.14 2.95 ± 0.09 4.07 ± 0.33 3.63 ± 0.03 3.52 ± 0.09
1 22.42 ± 0.25 20.01 ± 0.20 2.21 ± 0.07 2.63 ± 0.39 3.04 ± 0.21 2.40 ± 0.31
2 25.41 ± 0.20 23.68 ± 0.20 3.14 ± 0.05 4.58 ± 0.47 4.24 ± 0.33 3.82 ± 0.22
3 24.25 ± 0.90 21.20 ± 1.17 3.30 ± 0.20 4.22 ± 0.53 3.40 ± 0.70 3.34 ± 0.08
25 0 26.99 ± 0.31** 22.55 ± 1.21** 2.81 ± 0.16** 3.41 ± 0.31** 4.12 ± 0.36 3.97 ± 1.01
1 27.38 ± 0.29 26.13 ± 0.79 0.13 ± 0.14 -0.27 ± 0.36 3.28 ± 0.18 3.52 ± 0.22
2 28.52 ± 0.23 26.84 ± 0.35 -0.55 ± 0.13 -0.55 ± 0.28 3.32 ± 0.27 2.98 ± 0.15
3 28.32 ± 0.35** 26.81 ± 0.08** -0.29 ± 0.17** -0.47 ± 0.12** 3.12 ± 0.31 2.55 ± 0.48
45 0 26.38 ± 0.41** 22.90 ± 0.56** 2.99 ± 0.12** 3.62 ± 0.44** 3.61 ± 0.09 4.16 ± 0.45**
1 25.16 ± 0.84 23.40 ± 0.92 -0.14 ± 0.18 -0.58 ± 0.32 2.19 ± 0.74 1.57 ± 0.43
2 28.53 ± 0.59 27.25 ± 0.12 -0.52 ± 0.09 -0.35 ± 0.06 1.63 ± 0.37 2.03 ± 0.30
3 28.86 ± 0.14** 26.77 ± 0.42** -0.43 ± 0.06** -0.24 ± 0.05** 1.3 ± 0.19 2.04 ± 0.48**
pH5 4 0 25.71 ± 0.42** 25.46 ± 0.06** 2.98 ± 0.09** 2.47 ± 0.35 3.81 ± 0.24 3.88 ± 0.54
1 23.64 ± 0.44 21.59 ± 1.43 2.17 ± 0.12 2.17 ± 0.47 3.65 ± 0.14 3.36 ± 0.44
2 25.27 ± 0.35 26.75 ± 0.28 2.73 ± 0.19 2.89 ± 0.43 4.65 ± 0.35 4.25 ± 0.28
3 20.12 ± 0.58** 21.50 ± 1.07** 2.34 ± 0.10** 2.52 ± 0.26 3.79 ± 0.07 4.48 ± 0.21
25 0 26.31 ± 0.53 25.36 ± 0.24** 3.12 ± 0.15** 3.43 ± 0.17** 2.78 ± 0.36** 4.24 ± 0.21**
1 23.53 ± 0.44 27.27 ± 1.02 3.12 ± 0.56 -0.21 ± 0.13 2.13 ± 0.39 2.61 ± 0.19
2 27.49 ± 0.20 28.51 ± 0.38 -0.13 ± 0.25 -0.42 ± 0.13 2.78 ± 0.41 2.11 ± 0.24
3 26.32 ± 0.53 28.38 ± 0.36** -0.42 ± 0.01** -0.53 ± 0.05** 2.82 ± 0.88** 2.17 ± 0.24**
45 0 25.94 ± 1.62 26.02 ± 0.47** 3.51 ± 0.28** 1.60 ± 0.02** 2.48 ± 0.43** 3.76 ± 0.04**
1 27.57 ± 0.98 28.76 ± 0.35 -0.56 ± 0.01 -0.69 ± 0.08 1.93 ± 0.30 0.59 ± 0.20
2 28.11 ± 0.57 29.25 ± 0.35 -0.56 ± 0.06 -0.44 ± 0.08 1.93 ± 0.17 1.41 ± 0.28
3 28.29 ± 0.28 29.81 ± 0.07** -0.65 ± 0.14** -0.43 ± 0.06** 1.53 ± 0.09** 1.51 ± 0.20**
pH7 4 0 22.30 ± 1.58 21.86 ± 0.63 3.13 ± 0.28 2.45 ± 0.59 4.37 ± 0.61 4.37 ± 0.71
1 20.12 ± 1.15 21.79 ± 0.79 2.30 ± 0.43 2.51 ± 0.54 3.91 ± 0.55 4.29 ± 0.71
2 25.23 ± 0.42 26.38 ± 0.42 2.41 ± 0.33 3.17 ± 0.15 4.84 ± 0.09 4.73 ± 0.16
3 24.00 ± 0.29 23.73 ± 0.51 2.67 ± 0.33 2.91 ± 0.39 3.58 ± 0.21 3.11 ± 0.74
25 0 25.42 ± 1.02 26.58 ± 0.63 2.22 ± 0.16** 2.12 ± 0.25** 4.67 ± 0.61** 2.60 ± 0.47
1 25.20 ± 0.43 24.56 ± 1.35 1.16 ± 0.32 -0.35 ± 0.39 3.76 ± 0.56 1.63 ± 0.68
2 28.26 ± 0.38 28.36 ± 0.61 -0.54 ± 0.12 -0.73 ± 0.05 3.90 ± 0.28 1.02 ± 0.42
3 28.71 ± 0.93 28.35 ± 0.34 -1.03 ± 0.14** -0.72 ± 0.08** 3.37 ± 0.10** 1.43 ± 0.80
45 0 20.86 ± 0.34** 26.09 ± 1.15 2.63 ± 0.58** 1.99 ± 0.14** 4.20 ± 1.40 3.36 ± 0.36**
1 27.33 ± 0.51 27.17 ± 1.00 -0.67 ± 0.06 -0.82 ± 0.05 1.52 ± 0.05 1.54 ± 0.33
2 27.00 ± 0.33 27.91 ± 0.31 -0.18 ± 0.10 -0.53 ± 0.08 1.24 ± 0.2 1.10 ± 0.41
3 28.21 ± 0.11** 28.90 ± 0.97 -0.40 ± 0.22** -0.48 ± 0.04** 1.27 ± 0.09 1.81 ± 0.19**
pH9 4 0 24.65 ± 0.09** 24.19 ± 0.23 3.42 ± 0.23** 3.44 ± 0.23** 3.29 ± 0.08 5.29 ± 0.15**
1 22.19 ± 0.36 22.75 ± 0.49 2.72 ± 0.22 2.33 ± 0.26 2.98 ± 0.39 3.46 ± 0.39
2 23.67 ± 1.13 26.34 ± 0.40 3.48 ± 0.23 2.53 ± 0.11 4.36 ± 0.16 4.63 ± 0.25
3 21.18 ± 0.29** 23.99 ± 2.43** 2.57 ± 0.43** 2.68 ± 0.05** 3.34 ± 0.53 4.01 ± 0.39**
25 0 26.41 ± 0.17 21.88 ± 0.47** 3.89 ± 0.13** 2.44 ± 0.28** 3.59 ± 0.11** 4.03 ± 0.89**
1 27.24 ± 0.52 26.46 ± 0.49 -0.20 ± 0.36 -0.19 ± 0.29 2.42 ± 0.32 2.01 ± 0.58
2 29.02 ± 0.52 27.40 ± 0.37 -0.59 ± 0.13 -0.55 ± 0.08 2.15 ± 0.18 2.07 ± 0.57
3 27.78 ± 1.13 27.58 ± 0.44** -0.60 ± 0.07** -0.47 ± 0.18** 1.84 ± 0.27** 2.18 ± 0.43**
45 0 25.57 ± 0.38** 24.03 ± 0.23** 3.34 ± 0.06** 3.15 ± 0.10** 3.10 ± 0.13** 4.95 ± 0.26**
1 27.95 ± 0.55 26.70 ± 0.49 -0.74 ± 0.04 -0.55 ± 0.09 1.77 ± 0.18 2.61 ± 0.23
2 27.63 ± 0.20 27.50 ± 0.44 -0.38 ± 0.13 -0.13 ± 0.10 1.95 ± 0.26 2.43 ± 0.30
3 28.36 ± 0.19** 28.58 ± 0.31** -0.48 ± 0.05** -0.15 ± 0.07** 1.84 ± 0.08** 1.93 ± 0.21**
*L = lightness, -a = greenness, +a = redness, -b = blueness, +b = yellowness
**The colorant was changed significantly (p<0.05) comparing the colorant of the extract in week0 and week3

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Table 4 %inhibition of freeze dried and spray dried extract before and after stability test

Freeze dry Spray dry


Conditions

Before After Before After


4°C 25°C 45°C 4°C 25°C 45°C

Light 23.06 ± 1.74** 6.93 ± 2.83** 13.11 ± 1.34** 29.84 ± 3.15** 6.60 ± 2.74** 17.92 ± 3.00**

Dark 36.96 ± 0.83 23.15 ± 1.68** 21.17 ± 2.66** 36.87 ± 2.16** 22.07 ± 0.79** 24.75 ± 1.35**

pH3* 48.18 ± 5.80 25.74 ± 5.59 28.81 ± 0.73 51.30 ± 1.51 12.02 ± 0.88** 16.78 ± 4.85**
49.19 55.46
pH5* 30.58 ± 1.37** 12.47 ± 1.28** 17.46 ± 1.02** 39.37 ± 6.61 37.95 ± 1.70** 13.81 ± 0.91**

pH7* 24.91 ± 2.44** 7.90 ± 0.74** 17.42 ± 1.82** 27.62 ± 2.14** 8.99 ± 0.85** 14.18 ± 2.49**

pH9* 29.16 ± 1.37 8.67 ± 1.77** 12.43 ± 2.10** 28.31 ± 0.56** 11.02 ± 1.10** 12.68 ± 1.31**

* Determination under dark condition


*% inhibition was changed significantly (p<0.05) comparing before and after stability test

IC50 was an amount of antioxidant presented in sample 6. References


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Laboratory of Plant Cell Genetics Department
of Plant Molecular Biology Université de
Lausanne, CH 1015 Lausanne, Switzerland.

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