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

1
 Figure 1 The structure of betaxanthin, betelain and betacyanin
According to Jackman and Smith (1996), the
reduction with the closed structure of cyclo-Dopa
provides the electronic resonance to the diphenolic
aromatic ring. This extra conjugation shifts the
maximum absorption from 480 nm (yellow,
betaxanthins) to about 540 nm (violet, beacyanins).
Betalains replace the anthocyanins in most of the plants
in the order Caryophyllales (Strack, Vogt and
Schliemann, 2003). Betalain is sensitive to light, pH,
temperature and water activity (Stintzing & Carle,
2004) so its storage condition in solution form is
inappropriate (Azeredo, 2009) (Figure 1).
Accordingly, the popular methods to remove water
component from emulsion or suspension for a better
stability while storage are freeze drying and spray
drying. Spray drying is the atomizing of an emulsion or
suspension in a drying chamber with hot air circulation.
The solution is contacted with hot air which cause the
water. Spray drying has good character reconstitution,
low water activity which is suitable for transport and
storage (Khan et al., 2010). The advantages of spray
drying are low costs and short contact time (Sagar and
Kumar, 2010). Spray drying can generate sticky
products sometimes (Hennigs et al., 2001). Another
technique freeze drying, which forms frozen condition
of the solution followed by drying sublimation under
vacuum, and the solid directly changes into ice in vapor
without becoming liquid (Kumar et al., 2011).
According to the major composition of the extracts
from Beetroot as Betalains, it has been reported to be
antioxidant (Tomasz, 2016). Therefore, this extract
may posses as cosmetics ingredient for antioxidant and
color properties (Kapadia & Rao, 2012) . The purpose
of this study is to investigate and determine the effect
of light, pH and temperature on the color and
antioxidant activity from Beetroot extract by spray
drying and freeze drying for color additive
development in cosmetic.
2. Materials and Methods
2.1 Plant collection
The fresh beetroot was bought from convenient
store in Nakhonratchasima, Thailand. It was washed
with tap water and cut into 1cm x 1cm cube pieces. It
was stored at 4℃ until use.
2.2 Preparation of the plant extract
The 1.6 kg of Beetroot was used to extract by
maceration technique. The beetroot was soaked in 45%
ethanol at a ratio 1:3 (w/v) and incubated in incubator
shaker at 225 rpm, for 24 hours at 30 ℃ thrice to get
the most extract. The mixture was filtered by suction
filtration method and concentrated by a rotary
evaporator under vacuum at 50°C (Ravichandran et al.,
2013).
2.3 Chemical Substances
Ascorbic acid (Sigma-Aldrich, USA), Ethanol
(Sigma-Aldrich, USA), Hydrochloric acid 37%
(Sigma-Aldrich, USA), Sodium chloride (Sigma-
Aldrich, USA) were used in this study.
2.4 Reagents
2,2-diphenyl-1-picrylhydrazyl (Sigma-Aldrich,
USA) were purchased for this study.
2.5 Equipments
The 2 digits’ balance, and 4 digits’ balance (Ohaus,
USA), Beaker 50, 100, 250, 500 mL (Durex,
Germany), Dropper with Rubber (Pyrex, Germany),
Freeze dryer (Labconco, USA), Glass Funnel (Pyrex,
Germany), Incubator shakier (Sithiporn Associates
Co., Ltd, Thailand), Laborator bottle (Durex,
Germany), Micropipette (Thermo Fisher Scientific,
USA), Micropipette Tips (Thermo Fisher Scientific,
USA), Microplate Reader (BMG, Australia), Rotary
evaporator (Eyela,N-1000 from Sithiporn Associates
Co., Ltd, Thailand), Round Bottom Flask 500 mL
(Pyrex, Germany), Spectrophotometer (Sithiporn
Associates Co., Ltd, Thailand), Spray dryer (JCM Best
Technology Co., Ltd., Thailand), Test tube (Pyrex,
Germany), Test tube, Stirring rods (Pyrex ,Germany),
UV-Visible spectrophotometer (Biochrom, England),
Ultrascan VIS (Hunterlab, USA), Volumetric flasks
2
(Isolab, Germany), 96-well plate (Thermo Scientific)
were used in this study.
2.6 Drying methods
2.6.1 Freeze drying
Pre-freezing was performed by FreeZone Benchtop
Shell Freezer at -30°C. The sample was frozen by
FreeZone Benchtop Freeze Dryer for 5 days to ensure
complete drying. The pressure was reduced to 0.47
mBar and the temperature was reduced to -40°C or
lower. The final sample was stored in glass bottle and
covered with aluminium foil for later use.
2.6.2 Spray drying
According the the applied method described by
Bazaria et al. (2016), the sample was dried on a JCM
minilab spray dryer. The drying conditions were:
drying air inlet temperature of 200°C and outlet 80°C;
atomization pressure: 0.036 psi; average flow drying
air of 15 litres/hr. The final sample was stored in plastic
bottle and covered with aluminium foil for later use.
2.7 Spectrophotometric determination of Phenolic
compound in Beetroot Extract
The stock solution of Beetroot extract was prepared
at concentration of 4 mg/ml in DI water at 4°C and then
diluted into the concentration range of 31.25 to 4,000
µg/ml. The UV spectrophotometric determination of
the extracts was scanned at the wavelength of 200-600
nm.
2.9 Stability test
The 1 µg/ml sample solution in DI water was kept
in different conditions for 3 weeks. The stability of the
samples was measured by the colorant and the
antioxidant activity of the sample before and after
being kept in different conditions.
2.9.1 Stability test on light and temperature
According to the method described by Chandran et
al. (2012) and Woo et al. (2011), the effect of light and
temperature on colorant stability was done with 3 ml of
samples solution inside test tube covered with and
without aluminium foil, sealed with parafilm and kept
at three different temperatures: 4°C, 25°C and 45°C for
3 weeks. Fume hood, hot air oven, refrigerator and
water baht were used. The extract also was exposed for
heat-cold cycle for 6 cycles. (Molina, Hernández-
Martínez, Cortez-Valadez, García-Hernández &
Estevez, 2014).
2.9.2 Stability test on pH and temperature
According to the method described by Wong et al.
(2014), the effect of pH and temperature on colorant
stability was performed with 3 ml of samples solution
inside test tube prepared in four different pH: 3, 5, 7
and 9 covered with aluminium foil to avoid the effect
of light and kept at three different temperatures: 4°C,
25°C and 45°C for 3 weeks. Fume hood, hot air oven,
refrigerator and water baht were used.
2.9.3 Evaluation of color
The sample before and after being kept in different
conditions were evaluated using Ultrascan VIS in
terms of Hunter ‘L’ (lightness), ‘a’ (redness and
greenness) and ‘b’ (yellowness and blueness). The
instrument was calibrated with standard white and
black tiles. All the experiments were done in triplicates.
2.9.4 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
assay
According to the method described by Sasa et al.
(2012), antioxidant activity of Beetroot extract before
and after all different testing conditions were
determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
method with slightly modification of Straus et al.,
2012. The 0.1mM of DPPH solution was freshly
prepared in ethanol. 50 µl of the extract was added to
150 µL of 0.1 mM DPPH solution. The mixtures were
mixed and kept under dark condition at room
temperature for 30 min. The absorbance (ABS) was
measured by spectrophotometer at 520 nm. Ethanol
was used as a blank. All the experiments were done in
triplicates. The scavenging ability of the extract and
ascorbic acid toward DPPH radical was calculated by
ให้ ข้อคิดเห็น [U3]: I think this unit must be mg/ml rather than
using Scavenging effect equation %: (A0 – A1 ) x 100% ug/ml ka.
/A0, where A0 is absorbance of the control reaction and
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
calibration curve by plotting the extract concentration
versus the corresponding scavenging effect.
2.10 Statistical analysis
The IBM SPSS Statistics 22 program (trial version)
was used for data analysis. The data were analyzed and
compared by t-test. The level of statistical significance
was taken at p-value of less than 0.05 levels.
3. Results and Discussion
3.1 Extraction and Drying methods
ให้ ข้อคิดเห็น [U4]:
Beetroot was extracted by maceration, then was
dried by freeze drying and spray drying method.
The percentage yield of the crude extract was 3.057%.
ให้ ข้อคิดเห็น [U5]: เอาไปอยู้ใน Result and discussion
The extract has red color and sticky feeling. The extract
from freeze drying is less sticky than the one from
spray drying.
3.2 UV/Vis Spectrophotometry
According to research by Khoo et al. (2011)
including the report from Zryd et al. (2003) which
characterized the character of carotenoids,
betaxanthins and betacyanins under UV/Vis
spectrophotometer exhibited maximum absorption at
λmax between 330 - 350, 470 - 486 and 535 - 550 nm,
3
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

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

5
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

that decreases the original DPPH concentration by
50%. The lower value of IC50 has a higher antioxidant
activity. The beetroot extract inhibits free radical less
than ascorbic acid.
From table 4, it was observed that the extract from
freeze drying and spray drying at pH3, 4°C has almost
the same inhibition property with the extract before
stability test. After stability test, it has been concluded
that the best condition which stablilise the antioxidant
activity from beetroot extract is pH3 and 4°C.
4. Conclusion
In this study, the results show that beetroot extract
achieved by maceration technique and dried by freeze
drying and spray drying technique both have similar
colorant and colorant stability. The temperature plays a
huge role in affecting stability of the extract's color. As
the antioxidant activity, the extract has the potential to
be antioxidant which is useful for cosmetics
application. The best storage condition for the stability
of the color and antioxidant activity of the extract is
pH3 and 4°C. Therefore, extract could be used as the
colorant additive and active ingredient as antioxidant
in cosmetic and also can be used with synthetic
pigment in cosmetic for a better stability in color of the
product due to its unstable colorant with high
temperature.
5. Acknowledgements
The authors would like to express our sincere
gratitude to Dr. Tinnakorn Theansungnoen and people
who supporting this study. The authors would also like
to thanks the School of Cosmetic science, Mae Fah
Luang University for providing equipment and
chemical substance.
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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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