Antioxidant and Antiaging Assays of Hibiscus sabdariffa Extract and Its Compounds

Abstract

Skin aging is a complex biological process due to intrinsic and extrinsic factors. Free radical oxidative is one of extrinsic factors that induce activation of collagenase, elastase and hyaluronidase. Natural product from plants has been used as antioxidant and antiaging. This study aimed to evaluate antioxidant and antiaging properties of Hibiscus sabdariffa extract (HSE) and its compounds including myricetin, ascorbic acid, and β carotene. The phytochemical of H. sabdariffa was determined using modified Farnsworth method and presence of phenols, flavonoids and tannins were in moderate content, whereas triterpenoids and alkaloids were in low content. Total phenolic content performed using Folin-Ciocalteu method, was 23.85 μg GAE/mg. Quantitative analysis of myricetin, β-carotene, and ascorbic acid of HSE was performed with Ultra-High Performance Liquid Chromatography (UHPLC) that shows 78.23 μg/mg myricetin, 0.034 μg/mg β-carotene, whilst ascorbic acid was not detected. HSE has lower activity on DPPH (IC50 = 195.73 μg/mL) compared to β-carotene, the lowest in ABTS assay (IC50 = 74.58 μg/mL) and low activity in FRAP assay (46.24 μM Fe(II)/μg) compared to myricetin, β-carotene. Antiaging was measured through inhibitory activity of collagenase, elastase, and hyaluronidase. HSE had weakest collagenase inhibitory activity (IC50= 750.33 μg/mL), elastase inhibitory activity (103.83 μg/mL), hyaluronidase inhibitory activity (IC50 = 619.43 μg/mL) compared to myricetin, β-carotene, and ascorbic acid. HSE contain higher myricetin compared to β-carotene. HSE has moderate antioxidants and lowest antiaging activities. Myricetin is the most active both antioxidant and antiaging activities.

Full text

Natural Product Sciences
23(3) : 192-200 (2017)
https://doi.org/10.20307/nps.2017.23.3.192
192
Antioxidant and Antiaging Assays of Hibiscus sabdariffa Extract
and Its Compounds
Wahyu Widowati
1,
*, Andani Puspita Rani
1
, R. Amir Hamzah
1
, Seila Arumwardana
2
, Ervi Afifah
2
,
Hanna Sari W. Kusuma
2
, Dwi Davidson Rihibiha
2
, Hayatun Nufus
2
, and Annisa Amalia
2
1Medical Research Center, Faculty of Medicine, Maranatha Christian University,
Jl. Prof. Drg. Surya Sumantri No. 65 Bandung 40164, West Java, Indonesia
2Aretha Medika Utama, Biomolecular and Biomedical Research Center,
Jl. Babakan Jeruk 2, No. 9, Bandung 40163, West Java, Indonesia
Abstract – Skin aging is a complex biological process due to intrinsic and extrinsic factors. Free radical oxidative
is one of extrinsic factors that induce activation of collagenase, elastase and hyaluronidase. Natural product from
plants has been used as antioxidant and antiaging. This study aimed to evaluate antioxidant and antiaging
properties of Hibiscus sabdariffa extract (HSE) and its compounds including myricetin, ascorbic acid, and β
carotene. The phytochemical of H. sabdariffa was determined using modified Farnsworth method and presence of
phenols, flavonoids and tannins were in moderate content, whereas triterpenoids and alkaloids were in low
content. Total phenolic content performed using Folin-Ciocalteu method, was 23.85 µg GAE/mg. Quantitative
analysis of myricetin, β-carotene, and ascorbic acid of HSE was performed with Ultra-High Performance Liquid
Chromatography (UHPLC) that shows 78.23 µg/mg myricetin, 0.034 µg/mg β-carotene, whilst ascorbic acid was
not detected. HSE has lower activity on DPPH (IC50 = 195.73 µg/mL) compared to β-carotene, the lowest in
ABTS assay (IC50 = 74.58 µg/mL) and low activity in FRAP assay (46.24 µM Fe(II)/µg) compared to myricetin,
β-carotene. Antiaging was measured through inhibitory activity of collagenase, elastase, and hyaluronidase. HSE
had weakest collagenase inhibitory activity (IC50 = 750.33 µg/mL), elastase inhibitory activity (103.83 µg/mL),
hyaluronidase inhibitory activity (IC50 = 619.43 µg/mL) compared to myricetin, β-carotene, and ascorbic acid.
HSE contain higher myricetin compared to β-carotene. HSE has moderate antioxidants and lowest antiaging
activities. Myricetin is the most active both antioxidant and antiaging activities.
Keywords –Hibiscus sabdariffa, Myricetin, β-carotene, Ascorbic acid, Antioxidant, Antiaging
Introduction
The process of skin aging has been divided into two
categories: intrinsic and extrinsic aging.
1-3
Intrinsic skin
aging or natural aging is caused by changes in elasticity
of the skin over time, whilst extrinsic aging is caused by
environmental factors such as ultraviolet damage, pollution,
harsh weather, and cigarette smoke.
4
Extrinsic skin aging
is predominantly a result of exposure to solar radiation
(photoaging).
1-3,5
UV exposure causes physical changes to
the skin due to alterations that occur in the connective
tissue via the formation of lipid peroxides, cell contents
and enzymes.
6
Antiaging cosmetics are touted to erase wrinkles and
rejuvenate the skin. Most of these products serve only to
camouflage wrinkles and moisturize the skin.
7
These
characteristics of cosmetic are due to the presence of
synthetic or natural ingredients that diminish the exhibition
of free radicals in skin and manage the skin properties for
a long time.
8
The primary treatment of photoaging is
photoprotection, but secondary treatment could be
achieved with the use of antioxidants and some novel
compounds such as polyphenols.
9
Antioxidant treatment
may be an useful way to reduce the harmful effects of
reactive oxygen species and to protect skin from aging.
10
The antioxidant activity and Sun Protector Factor (SPF)
assay can be used to curb skin from photodamage.
Elastase, hyaluronidase, and collagenase have been
known as aging-related enzymes.
11
The elastase inhibition
can be used for determination of skin elasticity; furthermore,
collagenase has been required to inhibit for retention of
skin elasticity and tensile strength of the skin. Moreover,
*Author for correspondence
Wahyu Widowati, Faculty of Medicine, Maranatha Christian Uni-
versity, Bandung 40164, Indonesia.
Tel: +62-22-2017621; E-mail: wahyu_w60@yahoo.com

Vol. 23, No. 3, 2017 193
antiglycation assay checks the devastation and loss of
collagen, and hence it reduces wrinkle and aging from
skin. Hyaluronic acid holds the water together and keeps
the body smooth, watery and lubricated, so that hyaluro-
nidase inhibition assay can check the moisture present in
the body.
8
H. sabdariffa (Malvaceae), commonly known as roselle
is a medicinal plant that has been reported to be a good
source of antioxidants due to being rich in ascorbic acid,
myricetin and β-carotene.
12
H. sabdariffa is an annual
crop used in food, animal feed, nutraceuticals, cosme-
ceuticals and pharmaceuticals. The calyces, stems, and
leaves are acid in flavor. The juice from the calyces is
claimed to be a health-enhancing drink due to its high
content of ascorbic acid, anthocyanins and other antioxi-
dants.
13
In the present study, free radical scavenging
activities of H. sabdariffa extract (HSE) and myricetin, β-
carotene, and ascorbic acid (Fig. 1) were evaluated. Their
antiaging activity also were investigated by measuring
inhibitory activity toward enzymes collagenase, elastase,
and hyaluronidase.
Experimental
Preparation of H. sabdariffa extract −The plants of
H. sabdariffa were collected from Subang, West Java,
Indonesia. The plants were identified by herbarium staff,
Department of Biology, School of Life Science and
Technology, Bandung Institute of Technology, Bandung,
West Java, Indonesia. The dried flower of H. sabdariffa
(1600 g) were mashed, extracted using 500 mL destilled
ethanol 70% by a maceration method. Every 24 h the
ethanol filtrate was filtered and wastes were remacerated
until colourless filtrate. Maceration were concentrated
using 50
o
C evaporator to obtain extract. The extract of H.
sabdariffa (76.79 g) was stored at −20
o
C, ready used for
experiment.
14
Standard compounds used in this study
were myricetin [Sigma Aldrich M6760, USA] with 96%
purity, β-carotene with purity 98% [Biopurify Phytoche-
micals 14031820, China] and ascorbic acid with purity
99% [Sigma Aldrich A5960, USA] .
Qualitative phytochemical screening assay − Phytoche-
mical screening of HSE was evaluated using modified
Farnsworth method to identify qualitatively presence of
phenols, steroids/triterpenoids, saponins, tannins, terpenoids,
flavonoids, and alkaloids as listed below.
15-17
Phenols identification −HSE (10 mg) was placed on a
dropping plate, then 1% FeCl
3
[Merck 1.03861.0250, USA]
was added into the sample. The presence of phenols
indicated by color formation green/red/purple/blue/black.
15-17
Steroids/triterpenoids identification –HSE (10mg) was
placed on a dropping plate, and then soaked with acetate
acid until the sample was covered. One drop of absolute
sulphate acid (H
2
SO
4
) [Merck 109073, USA] was added
to the sample after 10 - 15 min. The color formation of
green/blue shows the presence of steroids while red/
orange sediment indicates the presence of triterpenoids.
15-17
Saponins identification – HSE (10 mg) was put into
the test tube with some water and boiled for 5 min,
shaken it vigorously. Saponins content was indicated by
persistence of froth on the surface.
15-17
Tannins identification – HSE (10 mg) was added with
2 mL of HCl 2N [Merck 1003171000, USA] in the test
tube, then heated on a water bath for 30 min. The mixture
was cooled down and filtered, the filtrate was added with
amyl alcohol [Merck 10979, USA]. Purple colour formation
indicates positive reaction for tannins.
15-17
Terpenoids identification – HSE (10 mg) was added
into a dropping plate, then vanillin and H
2
SO
4
was added
into the sample. Formation purple color on the mixture
shows presence of terpenoid content.
15-17
Flavonoids identification – HSE (10 mg) was added
into a test tube, and added Mg [Merck EM105815, USA]
and HCl 2N. The mixture sample was heated for 5 to 10
min, then it was cooled down and filtered, and then amyl
alcohol was added into it. The positive reaction was
shown by the formation of red or orange color.
15-17
Alkaloids identification – HSE (10 mg) was added into
a test tube, then 10% ammonia was added into the
sample. Chloroform was added to the mixture, forming
two layers of liquid and the bottom layer was collected.
HCl 1N was added to the liquid, forming two layers and
the upper layer collected and added with 1-2 drops of
draggendorf solution. The presence of yellow color indicated
the positive result.
15-17
Total phenolic content −Total phenolic was evaluated
Fig. 1. Chemical structure of compounds in the H. sabdariffa. (1)
Myricetin (C
15
H
10
O
8
), (2) Ascorbic acid (C
6
H
8
O
6
), (3) β-carotene
(C
40
H
56
).

194 Natural Product Sciences
using a modified colorimetric method by Widowati et al.
(2015).
18,19
The method involves the reduction of Folin-
Ciocalteu reagent (Merck 1.09001.0500, USA) by phenolic
compounds, with a concomitant formation of a blue
complex. The amount of total polyphenol is calculated
using the gallic acid calibration curve. The results are
expressed as gallic acid equivalent mg/100 mL of the
sample.
18-19
UHPLC – Quantification of HSE was performed with
LC-MS/MS (Accella 1250, Thermo Scientific) using
standard myricetin, β-carotene, and ascorbic acid. Samples
were dissolved in 1 mL methanol gradient grade and
filtered through 0.2 µm syringe. Formic acid 0.1% was
used as mobile phase. Absorbance was measured with
MS/MS Triple Q (quadrupole) mass spectrophotometer
TSQ Quantum Access MAX Triple Quadrupole (Thermo
Scientific) Electrospray Ionization (ESI) (voltage 3 kV;
evaporation temperature 250
o
C; capilary temperature
300
o
C; nitrogen 40 psi, and Aux 10 psi with argon gas)
controlled by software TSQ Tune operated with positive
palarity.
20
2,2-Diphenyl-1-picrylhydrazil (DPPH) assay −The
DPPH assay was conducted using the method from
Widowati et al.(2015) study.
18-19
The method is based on
the reduction of alcoholic DPPH solution in the presence
of a hydrogen-donating antioxidant due to the formation
of the non-radical from DPPH-H by the reaction.
21
Briefly,
50 µL samples, was added to each well in a 96-well
microplate. It was then followed by addition of 200 µL of
2,2-Diphenyl-1-picrylhydrazil (DPPH) [Sigma Aldrich
D9132, USA] solution (0.077 mmol/L in methanol) into
the well. The mixture then incubated in the dark for
30 min at room temperature. Afterwards, the absorbance
was read using a microplate reader (Multiskan™ GO
Microplate Spectrophotometer, Thermo Scientific, USA) at
517 nm wavelength. The DPPH extract and compounds
generally fades purple colour into a colourless when
antioxidant molecules quench DPPH free radicals.
14,18,19,22
The radical scavenging activity was measured using the
following formula :
Scavenging % = (Ac – As) / Ac ×100
Ac: negative control absorbance (without sample)
As: sample absorbance
The median inhibitory concentration (IC
50
) value of
DPPH activity were calculated.
14,18-19
ABTS-reducing activity assay −The antioxidant activity
of HSE, myricetin, β-carotene, and ascorbic acid were
measured using 2,2'-Azino-bis (3-ethylbenzothiazoline-6-
sulphonic acid)(ABTS
•+
) [Sigma Aldrich A1888-2G, USA]
diammonium salt free radical assay.
23
ABTS
•+
solution
was produced by reacting 14 mM ABTS and 4.9 mM
potassium persulfate [Merck EM105091, USA] (1:1 volume
ratio) for 16 h in dark condition at room temperature, then
the mixture was diluted with 5.5 mM PBS (pH 7.4) until
the absorbance of the solution was 0.70 ±0.02 at
wavelength 745 nm. In brief, 2 µL of samples were added
to each well at 96-well microplate, then to the samples the
fresh 198 µL ABTS
•+
solution were added. The absorbance
was measured at 745 nm after the plate incubated for 6
min at 30
o
C. The percentage inhibition of ABTS radical
(%) was determined by the ratio of reducing of ABTS
•+
absorbance in the presence of the sample relative to the
absorbance in the absence of the sample (negative
control). The median inhibitory concentration (IC
50
) were
also calculated.
23
FRAP assay −The ferric reducing antioxidant power
assay (FRAP) was estimated using modified method from
Mishra et al. (2006) and Widowati et al. (2014) studies.
23,24
The FRAP reagent was prepared freshly by mixing
10 mL of acetate buffer 300 mM (pH 3.6 adjusted with
addition of acetic acid), 1 mL of ferric chloride hexahydrate
[Merck 1.03943.0250, USA] 20 mM dissolved in distilled
water, and 1 mL of 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ)
[Sigma 3682-35-7, USA] 10 mM dissolved in HCl
40 mM. In 96-well microplate, 7.5 µL of samples were
mixed with 142.5 µL FRAP reagent then incubated for 30
min at 37
o
C. The absorbance value was measured at 593
nm with a microplate reader (Multiskan™ GO Microplate
Spectrophotometer, Thermo Scientific, USA). The standard
curve was made using FeSO
4
, between 0.019 and 95 μg/
mL FeSO
4
. The results of samples were expressed in µM
Fe (II)/µg extract.
23
Collagenase Assay −Collagenase inhibitory activity
was measured according to modified method of Sigma
Aldrich and Thring et al. (2009).
11,2 5,2 6
Mixed solution
included 10 µL Collagenase from Clostridium histolyticum
[Sigma Aldrich C8051, USA] (0.01 U/mL in the cool
aquades), 60 µL Tricine buffer (50 mM, pH 7.5, content
of 10 mM CaCl
2
dan 400 mM NaCl), 30 µL sample (0 -
250 µg/mL in the DMSO), then incubated at 37
o
C for 20
min. After incubated added 20 µL N-[3-(2-Furyl)acryloyl]-
leu-gly-Pro-Ala [Sigma Adrich F5135, USA] (1 mM in
the Tricine buffer) substrate. Absorbance was measured at
335 nm wavelength.
Inhibition activity = (1 −B/A)×100%
A = Absorbance sample test
B = Absorbance control

Vol. 23, No. 3, 2017 195
The median inhibitory concentration (IC
50
) of collagenase
assay were also calculated.
Elastase Assay −Elastase inhibitory activity was
measured by modified method of Sigma-Aldrich and
Thring et al. (2009).
11,25,26
Sample 10 µL (0 - 66.67 µg/
mL) was pre-incubated for 15 min at 25
o
C with 5 µL
elastase from porcine pancreas [Sigma Aldrich 45124,
USA] (0.5 mU/mL in the cool aquades), 125 µL Tris
buffer (100 mM, pH 8). Mixed solution was added 10 µL
N-Sucanyl-Ala-Ala-Ala-p-Nitroanilide substrate [Sigma
Aldrich 54760, USA] (2 mg/mL inTris buffer), and then
incubated for 15 min at 25
o
C. Absorbance was measured
by 410 nm wavelength.
Inhibition Activity = (1 −B/A)×100%
A = Sample absorbance
B = Control absorbance
The median inhibitory concentration (IC
50
) of elastase
assay were also calculated.
Hyaluronidase Assay −Hyaluronidase inhibitory activity
was measured by modified method of Sigma-Aldrich and
Tu & Tawata (2015).
25,26
Sample 25 µL (0 -166.67 µg/
mL) was pre-incubated for 10 min at 37
o
C with 3 µL
hyaluronidase from bovine testes type I-S [Sigma Aldrich
H3506, USA] (0.4 U/mL in the 20 mM phospate buffer,
pH 7 in the 77 mM sodium chloride and 0.01% bovine
serum albumin), and added 12 µL phospate buffer
(300 mM, pH 5.35) for 10 min at 37
o
C. And then, 10 µL
hyaluronic acid substrate (0.03% in the 300 mM phospate
buffer, pH 5.35) [Sigma Aldrich H5542, USA], and
incubated for 45 min at 37
o
C. Reaction decomposition of
hyaluronic acid stopping by added 100 µL acidic albumin
acid (24 mM sodium acetate, 79 mM acetate acid and
0.1% BSA). Mixed solution was incubated at room tem-
perature for 10 min, then absorbance was measured at
600 nm wavelength.
Quantification of inhibition activity by formula:
Inhibition activity = (1 −B/A)×100%
A = Sample absorbance
B = Control absorbance
The median inhibitory concentration (IC
50
) of hyaluro-
nidase assay were also calculated.
Stastical Analysis −Statistical analysis was conducted
using SPSS software (version 20.0). Value were presented
as Mean ±Standard Deviation. Significant differences
between the groups were determined using the Analysis
of variance (One Way ANOVA) followed by Tukey’s
HSD Post-hoc Test.
Results and Discussion
Calyces of H. sabdariffa has been known to posess
high antioxidants due its high content of ascorbic acid,
anthocyanins and other compounds such as myricetin and
β-carotene.
13
The present study showed the phytoche-
micals in HSE were phenols, flavonoids, and tannins in
moderate level (++), triterpenoids and alkaloids in low
content (+), whilst steroids, terpenoids, and saponins were
not detected (−) (Table 1).
The total phenolic content was calculated by using the
gallic acid calibration curve.
18,19
Total phenolic content in
the HSE was 23.85 ±1.17 µg GAE/mg (Table 2). Previous
study showed H. sabdariffa ethanol extracts achieved
total polyphenol content between 669.48 and 5012.54 mg
GAE 100/g.
27
Fig. 2, shows presence of three standard compounds in
extract as indicated by retention time. Quantitative
analysis was then measured (Table 3) in which HSE
contain myricetin (78.23 ng/mg), β-caroten, (0.034 ng/
mg) and ascorbic acid (<LOQ). Ascorbic acid was below
limit of detection which means that the con- centration
of ascorbic acid was very low in HSE. This result was not
consistent with previous research that the flower of H.
sabdariffa contained β-carotene, phytosterol, citric acid,
ascorbic acid, maleic acid, malic acid, niacin, pectin.
28
Antioxidants have been studied for their effectiveness
Table 1 . The result of qualitative phytochemical screening of
HSE (phenols, steroids/triterpenoids, terpenoids, saponins, flavonoids,
tannins and alkaloids)
Phytochemical content HSE
Phenols ++
Steroids/Triterpenoids −/+
Terpenoids −
Saponins −
Flavonoids ++
Tanins ++
Alkaloids +
++++: very high content; +++ : high content; ++ : moderate con-
tent; + : low content; − : not detected
Tab l e 2 . Total phenolic content of HSE with gallic acid standard.
Sample Linear Equation R
2
Total phenolic content
(µg GAE/mg)
HSE y = 0.003x + 0.024 0.998 23.85 ±1.17
The phenolic content of HSE was measured in triplicate.
In triplicate. Linear equation (Y = 0.003x + 0.024), coefficient of
regression (R
2
= 0.998) of Gallic Acid was calculated. The data
was presented as mean ±standard deviation.

196 Natural Product Sciences
Fig. 2. Chromatogram of UHPLC Analysis. (1) HSE and myricetin, (2) HSE and β-carotene, (3) HSE and ascorbic acid.
Table 3 . UHPLC analysis of compounds contained in HSE, quantification of HSE was performed with LC-MS/MS using myricetin, β-
carotene, and ascorbic acid as standard. The UHPLC analysis were performed in duplicate.
No Compounds Weight (g) Area Measured-Content (μg/mL) Calculated-Content (μg/mL)
1Myricetin 1.40 1,027 21.91 78.23
2β-carotene 1.80 8,817 0.361 0.034
3Ascorbic Acid 1.40 −0.01 < LOQ

Vol. 23, No. 3, 2017 197
in reducing the deleterious effects of aging and age-
related diseases.
29
DPPH is very stable free radical and
has advantage of being unaffected by side reactions, such
as metal ion chelation and enzyme inhibition.
30
In the
present study, HSE has low DPPH scavenging activity
(IC
50
= 195.73 µg/mL) compared to ascorbic acid (IC
50
=
33.54 µM or 5.91 µg/mL) (Table 4), while IC
50
of DPPH
activity of myricetin was 4.50 µg/mL as measured in our
previous study.
19
Myricetin, one of flavonoid compounds,
showed highest activitiy among treatments. This is supported
by the other study, that flavonoids can scavenge the
DPPH radical in a dose-dependent manner, and the DPPH
radical scavenging activity (IC
50
) was decreased in the
following order: myricetin (4 µM) > quercetin (5 µM) =
luteolin (5 µM) > kaempferol (12 µM) > chrysin > apigenin.
31
In this study, ascorbic acid also exhibited good scavenging
activity, which is validated by other study that ascorbic
acid can reduce the generation of reactive oxygen species
in some experimental models.
32-33
HSE showed the lowest ABTS-reducing activity (IC
50
=
74.58 µg/mL) compared to myricetin (IC
50
= 3.17 µM or
1.01 µg/mL), ascorbic acid (IC
50
= 27.38 µM, or 4.82 µg/
mL), β-carotene (IC
50
= 69.66 µM, or 37.40 µg/mL) (Table
5). These results indicate HSE has weak antioxidant
activity compared to three compounds. Myricetin has
greater antiradical activity than other flavonoids, and it
scavenges oxygen radicals and inhibits lipid peroxidation.
34
Myricetin can also act as a free radical scavenger when
keratinocytes experience UVB-induced damage.
35
Flavonols
that contain more hydroxyl groups (one to six OH groups)
such as myricetin, have higher free radical and superoxide
anion radical scavenging abilities. Myricetin is able to
rapidly donate a hydrogen atom to the radicals.
31
The present study showed that FRAP activity in
concentration-dependent manner, in which higher con-
centration increased FRAP activity. HSE at the highest
concentration (100.00 μg/mL) has moderate activity 46.24
µM Fe(II)/µg, compared to other compounds, myricetin at
the highest concentration (3.98 µg/mL) had reduced ferric
of 400.81 µM Fe(II)/µg, the highest concentration of β-
carotene (6.71 µg/mL) was 10.70 µM Fe(II)/µg, at highest
concentration of ascorbic acid (2.10 µg/mL) had value of
288.29 µM Fe(II)/µg (Table 6). HSE has lower antioxidant
activity in the FRAP assay compared to myricetin and
ascorbic acid. The moderate phenolic and flavonoid
content in HSE might be related to its antioxidant activity
(Table 1) and contained low myricetin according the
UHPLC analysis (Table 3). Another study showed that
flavonoids, including myricetin, exhibited antioxidant
properties against different types of free radicals.
36
Myricetin
as flavonoids has greater antiradical activity than other
flavonoids, that scavenges oxygen radicals and inhibits
lipid peroxidation.
37
Myricetin also has effect of antioxi-
dative and collagenase inhibitory activity caused Ultra
violet-A (UV-A) in human dermal fibroblasts depends on
the number and order of OH groups in those structure
compounds.
31
The combination of ascorbic acid and
vitamin E (α-tocopherol) was reported to synergistically
Table 4 . DPPH scavenging activity of HSE, myricetin, β-carotene, and ascorbic acid.
Samples The highest activity of ABTS
reducing activity (%) IC
50
(µg/mL) IC
50
(µM)
HSE 76.79 195.73 ±18.63 −
β-carotene 48.65 222.95 ±9.62 415.27 ±17.91
Ascorbic Acid 95.94 555.91 ±0.66 533.54 ±3.77
The data was presented as median inhibition concentration (IC
50
). The DPPH scavenging activity were measured in triplicate for each con-
centration of samples. Each DPPH scavenging activity of samples were calculated the IC
50
and each sample had three value of IC
50
. The
IC
50
value were presented as mean ±standard deviation, the IC
50
value were presented only µg/mL for HSE, both µg/mL and µM for β-
carotene, ascorbic acid.
Table 5 . ABTS-reducing activity of HSE, myricetin, β-carotene, and ascorbic acid were presented as IC
50
.
Samples The highest activity of ABTS
reducing activity (%) IC
50
(µg/mL) IC
50
(µM)
HSE 34.37 74.58 ±2.97 −
Myricetin 75.66 51.01 ±0.02 53.17 ±0.06
β-carotene 37.30 37.40 ±6.69 69.66 ±12.47
Ascorbic Acid 88.33 54.82 ±1.19 27.38 ±6.76
The ABTS-reducing activity were measured in triplicate for each concentration of samples. Each ABTS-reducing activity of samples were
calculated the IC
50
and each sample had three value of IC
50
. The IC
50
value were presented as mean ±standard deviation.

198 Natural Product Sciences
enhance free radical scavenging activity induced by UV
in human skin.
38-39
Ascorbic acid may play a key role in
the antioxidant and antiwrinkle effects of the mixture.
40
Ultra violet (UV) exposure causes physical changes of
the skin through complex pathways and finally generates
reactive oxygen species (ROS), matrix metalloproteinases
(MMPs) and elastase secretion,
41
which require antioxidant
to inhibit collagenase and elastase secretion. Collagenase
is responsible for extracellular matrix (ECM),
42
that
inhibit retention of skin elasticity and tensile strength of
the skin.
43
The present study showed that HSE has low
activity in collagenase assay (17.24%) compared to
myricetin (99.31%) and was comparable with ascorbic acid
(27.10%) and β-carotene (11.42%) at the highest
concentration (Fig. 2.A). The most active of collagenase
was myricetin with IC
50
value 36.60 µM or 11.65 µg/mL,
meanwhile the IC
50
of ascorbic acid was 465.67 µM or
82.01 µg/mL, HSE was 750.33 µg/mL, and β-carotene
was 1,369.50 µM or 734.22 µg/mL (Table 7). The result of
present study showed moderate collagenase inhibitory
Table 6 . FRAP activity of HSE, myricetin, β-carotene, and ascorbic acid.
Concentrations
HSE
(µg/mL)
HSE
FRAP Activity
(µM Fe(II)/µg)
Concetrations
Myricetin
(µg/mL)
Myricetin
FRAP Activity
(µM Fe(II)/µg)
Concetrations
β-carotene
(µg/mL)
β-carotene
FRAP Activity
(µM Fe(II)/µg)
Concetrations
Ascorbic acid
(µg/mL)
Ascorbic Acid
FRAP Activity
(µM Fe(II)/µg)
100.00 46.24 ±0.13
a
3.98 400.81 ±4.10
a
6.71 10.70 ±0.24
a
2.10 288.29 ±9.49
a
50.00 21.84 ±0.60
b
1.99 328.53 ±4.19
b
3.36 4.27 ±0.03
b
1.10 139.52 ±5.99
b
25.00 58.61 ±0.20
c
0.99 169.77 ±17.72
c
1.68 1.07 ±0.13
c
0.55 65.44 ±1.83
c
12.50 52.66 ±0.34
d
0.50 587.86 ±11.40
d
0.84 −1.28 ±0.64
d
0.28 32.25 ±1.84
d
The data was presented as mean ±standard deviation. The FRAP activity were measured in triplicate for each concentration of samples.
The concentrations level of samples were expressed µg/mL for HSE, µg/mL and µM for compounds (myricetin, β-carotene, ascorbic
acid). Results of FRAP activity were expressed in μM Fe(II)/μg sample. Different superscripts in the same column (
a, b, c, d
) indicate signifi-
cant differences among the means of groups (concentrations of HSE, myricetin, β-carotene, ascorbic acid) based on Tukey’s HSD Post-
hoc comparisons (P < 0.05).
Fig. 3. Effect of HSE (µg/mL), myricetin, β-carotene, ascorbic acid (µM) on collagenase, elastase, and hyaluronidase inhibitory activity.
(1) Collagenase inhibitory activity, (2) Elastase inhibitory activity, (3) Hyaluronidase inhibitory activity.

Vol. 23, No. 3, 2017 199
activity of HSE compared to other compounds. However,
myricetin exhibited the highest collagenase inhibitory
activity. As reported in previous study, myricetin inhibited
the wrinkle formation formation in UVB irradiation in
mouse skin, that might block damage to the basement
membranes by inhibiting expression and activity of
MMP-9.
44
Elastin, an ECM protein, is most abundant in organs
that provides elasticity to the connective tissues. Therefore,
inhibition of the elastase activity ingredients could be
used in cosmetic formulation to protect against skin aging
and wrinkles.
8
Myricetin exhibited the most active with
the highest elastase inhibitory activity 92.86% and IC
50
value 1.20 µg/mL or 3.76 µM. HSE exhibited the highest
elastase inhibitory activity IC
50
value 103.83 µg/mL,
meanwhile the IC
50
value of ascorbic acid was 15.47 µg/
mL or 87.83 µM and the IC
50
value of β-carotene was
79.95 µg/mL or 148.92 µM and (Fig. 2B, Table 7). These
findings were supported with previous study of flavonoid
effects on elastase release at the final concentration range
of 0.5 to 10 μmol/L. Myricetin inhibited elastase release at
the highest concentration tested (10 μmol/L).
45
Hyaluronidase inhibitory was determined by measuring
the amount of N-acetylglucosamine splited from sodium
hyaluronate.
46
HSE has highest IC
50
value (619.43 µg/
mL) compared to ascorbic acid (160.58 µM or 28.28 µg/
mL), β-carotene (34.21 µM or 18.35 µg/mL) and myricetin
(21.30 µM or 6.78 µg/mL). Myricetin has highest antiaging
activity of all treatments. Myricetin might strongly block
damage in basement membranes that can prevent UVB-
induced wrinkle formation. β-carotene also plays an
important role in the aging process as a mediator in redox
processes.
47
It has been found that ascorbic acid and β-
carotene are potent as radiation protectors.
48
Ascorbic acid
and β-carotene can act as antiaging compounds and
protect cell membrane from oxidative stress.
47
In vivo
study in pig skin demonstrated that the combination of
topical ascorbic acid and vitamin E synergistically provide
protection against UV-induced erythema and formation of
sunburn cells and thymine dimers.
49-50
Ascorbic acid, vitamin
E and polyphenols were reported to have significant
antiaging effects.
51
Those compounds have potency to
inhibit hyaluronidase by lowering its viscosity and
increasing the permeability.
52
Acknowledgements
This research was supported by Biomolecular and
Biomedical Research Center, Aretha Medika Utama,
Bandung, Indonesia for research grant, laboratory facilities
and research methodology.
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Received February 9, 2017
Revised June 7, 2017
Accepted June 8, 2017