Reverse UVB-induced photoaging by Hibiscus sabdariffa calyx

aqueous extract

Jian Li,a† Yi-Ru Lu,b† I-Fan Lin,c Wenyi Kang,d Hong-bin Chen,e Hsu-Feng Lu,f* Hui-Min
Accepted Article
David Wanga,e,g,h,i*

a
College of Food and Biological Engineering, Jimei University, Xiamen, 361021, China

b
Department of Bachelor Program of Biotechnology, National Chung Hsing University,

Taichung City 402, Taiwan

c
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402,

Taiwan

d
Joint International Research Laboratory of Food & Medicine Resource Function, Henan

Province, Henan University, Kaifeng 475004, China

e
College of Oceanology and Food Science, Quanzhou Normal University, Quanzhou

362000, China

f
Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei 112, Taiwan

g
Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung

City 402, Taiwan

h
College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan,

This article has been accepted for publication and undergone full peer review but
has not been through the copyediting, typesetting, pagination and proofreading
process which may lead to differences between this version and the Version of
Record. Please cite this article as doi: 10.1002/jsfa.10063
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 i
Department of Medical Laboratory Science and Biotechnology, China Medical University,

Taichung City 404, Taiwan

†
These authors contributed equally to this work.
Accepted Article

* Corresponding author:

Hsu-Feng Lu, Ph.D.

Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei 112, Taiwan
Mobil: 886-953655645
E-mail: luxufeng77@gmail.com

Hui-Min David Wang, Ph.D.

Professor, Graduate Institute of Biomedical Engineering, National Chung Hsing University,
Taichung City 402, Taiwan
Mobil: 886-935753718
TEL: 886-4-22840733#651
E-mail: davidw@dragon.nchu.edu.tw
https://sites.google.com/site/davidbiocosme/home

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 Abstract
BACKGROUND: Hibiscus sabdariffa is popularly used in daily life, and its extract is widely
applied in food and cosmetics. However, it has not been evaluated for its anti-aging effects in
academic fields yet.

RESULTS: H. sabdariffa calyx aqueous extract (HSCAE) have shown potential activities on
Accepted Article

collagenase activity suppression, tyrosinase activity inhibition, and anti-oxidation as a free

radical scavenger. The current investigation presented that HSCAE was not cytotoxic in skin
fibroblasts, and it significantly decreased ultraviolet B (UVB)-induced reactive oxygen
species (ROS) on a flow cytometry assay. Moreover, HSCAE reduced matrix
metalloproteinase (MMP) expressions, increased tissue inhibitor of metalloproteinase
(TIMP)-1 level, and enhanced collagen contents by inhibiting collagenase activity. It also
blocked mRNA and protein expressions of melanin production pathway key factors,
including microphthalmia-associated transcription factor (MITF), tyrosinase,
tyrosinase-related protein-1 (TRP-1) and dopachrome tautomerase-2 (TRP-2).

CONCLUSION: These results demonstrated for the first time the potential of HSCAE as a
natural antioxidant with the ability to maintain collagen production and to decrease melanin
syntheses under UVB radiation, for anti-aging.

Keywords: Hibiscus sabdariffa, antioxidant, anti-aging, collagen, melanin

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 INTRODUCTION

Skin is the largest organ of the body as the first defense against physical and chemical

damages, and microbial infections. In the physiological functions, it plays important roles in

retaining water, secreting sweat, sensing pain, regenerating dermal tissues and regulating
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body temperature1. Human skin is comprised of three layers, namely epidermises, dermis,

and hypodermis. The epidermis locates on the outermost surface, and the hypodermis is in

the bottom part. The dermis includes elastic fibers, collagen, and reticular fibers, and it

contributes extracellular matrix (ECM) to support cell proliferation; elastic fibers and collagen

fibers account for 2% and 90% of the skin, respectively. The exposure to a plethora of

oxidative stress on the skin causes damage to collagen and elastin. The oxidative stress is

produced by ultraviolet (UV) radiation, free radicals and reactive oxygen species (ROS) in

both external environments and internal resources2. Overproduction of ROS can induce

oxidative stress, resulting in cells failing to maintain normal physiological redox-regulated

functions3. Therefore, anti-oxidants are essential for human, for their ability to scavenge free

radicals and reduce the abnormal physiological degradations4.

As UV radiation is ubiquitously and naturally emitted from the sunshine, almost each

person is exposed to UV with a daily basis, and the UV light is essential for ordinary

physiological bio-functions. Nevertheless, during times of environmental stresses, such

as UVB or heat exposure, ROS level increases severely to result in noteworthy damage to

cellular functions and structures. In the current studies, it was demonstrated that the

accumulation of ROS decreased organism salubrity because the oxidative damage was a

contributor to the senescence1,2. Aging is a natural process for everyone and evoking

various diseases, and skin is highly responsive to photo-aging due to oxidative stress

created by ROS5. ROS induced from UVB damage on cellular DNA, RNA, proteins, and

lipids, which in theory, contributed to the physiology of aging. Due to skin aging caused by

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 internal and external factors, most people have more than one skin disorder after 65 years

old. Skin aging leads to dry or thin skin, unpleasant spots and wrinkles; in addition, loss of

subcutaneous tissue from the skin would lead to sunken cheeks and eye socket. The action

of skin aging can briefly divide into three categories: ROS, decreasing replication ability of
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cells and degradation of ECM. ROS is usually produced as side products of aerobic

metabolism in the mitochondria, causing intrinsic aging6. Cellular senescence is the

decrease of cell replication ability with time, often occurs in the aged skin. With time, the

increased expression of matrix metalloproteinase (MMP), which degrades collagen in ECM,

has been shown in senescent fibroblasts4. Rising tissue inhibitor of metalloproteinase (TIMP)

prevents collagen degradation by MMP and has a beneficial effect on keeping the collagen

content in the skin7.

Melanin provides pigments for our skin, hair and eyes, serving as a protection for cellular

organelles from UV rays. However, most people don’t like the pigment accumulation and

darken-skin for their appearances. Melanin is produced by melanocytes that situate at the

stratum basale in the skin and as the final product of melanogenesis from the substrate of

tyrosine8. The synthesis of melanin is associated with microphthalmia-associated

transcription factor (MITF) and three essential enzymes: tyrosinase, tyrosinase-related

protein (TRP-1) and Dopachrome tautomerase (TRP-2). Tyrosinase transforms L-tyrosine

into L ‑3,4‑dihydroxyphenylalanine (L ‑DOPA); L ‑DOPA could be converted into DOPA

Quinone; DOPA Quinone is turned into DOPAchrome; and TRP-1 and TRP-2 then

metabolize DOPAchrome into eumelanin and pheomelanin9-10. The inhibition of tyrosinase

and related protein expressions and activities is a successful strategy in in reducing melanin

production.

Hibiscus sabdariffa is native to West Africa, India, and Malaysia, and implemented in

several aspects. Its stem applies to cultivate for phloem fiber, and the red calyces are used

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 as a natural colorant11. The edible calyces can be made into jam, jelly, sauce and wine; its

seeds contain high protein and are used as a food ingredient in Africa12. Bioactive

ingredients of H. sabdariffa, such as anthocyanins, anthocyanidins with a sugar moiety,

gallic acid, syringic acid, caffeic acid and flavonoids, have been wildly studied for their
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antimicrobial, plasma cholesterol reducing and immune-regulating functions13-16. Studies

also reported extracts from Hibiscus sabdariffa inhibits cancer cells metastasis17-18. It is

known that flavonoids inhibit oxidative stress in vitro by acting either as free radical

scavengers or as metal chelating agents19. However, none of the studies to date have

investigated the anti-aging effect of H. sabdariffa extract. Therefore, in our study, we

examined the anti-oxidative capacities, photo damage repair, and anti-melanin production of

H. sabdariffa calyx aqueous extract (HSCAE), and as we know, it is the first time to be

investigated in anti-photoaging effects.

MATERIALS AND METHORDS

Reagents and materials

Vitamin C (L-ascorbic acid), dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl

(DPPH), ethylenediaminetetraacetic acid (EDTA), potassium ferricyanide, ferric chloride,

FeCl2·4H2O, trizol reagent, 1-bromo-3-chloropropane (BCP), 3-(4,5-dimethylthiazol-2-yl)

-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM) and

butylated hydroxyanisole (BHA) were purchased from Sigma-Aldrich Chemical Corp. (St.

Louis, MO, USA). Fetal bovine serum (FBS) and antibiotics were purchased from Gibco

Corp. (MA, USA). TOOLS 2  SYBR quantitative reverse transcription polymerase chain

reaction (qRT-PCR) Mix was bought from Biotools (Taipei, Taiwan). Collagenase Activity

Colorimetric Assay Kit was acquired from BioVision (San Francisco, CA, USA). HSCAE was

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 extracted with cold water at 4 ℃ for 24 hours, followed by vacuum concentration,

lyophilization, and pulverization to make a water-soluble powder; 0.1 g extract was dissolved

in 1 mL water, followed by 0.22 µm filtration.

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DPPH free radicals scavenging capacity assay

Antioxidant activity was measured by free radical scavenging and hydrogen donation20. In

brief, a 0.25 mmol L-1 DPPH solution in methanol was prepared, and 99 μL of DPPH solution

was added to a 1 μL of each material sample solution. After 30 min, the absorption at OD517

nm was measured. L-ascorbic acid at 100 μmol L-1 was used as the positive control. The

inhibitory effect was obtained based on the following equation:

(1)

Ferrous ion chelating activity assay

Ferrous ion chelating activity is measured by a metal scavenging outcome, as a measure of

anti-oxidation ability21. In brief, 69 μL of methanol and 10 μL of 2 mmol L-1 ferrous chloride

solutions were added to a 1 μL of 10 mmol L-1 material sample solution, then mixed with 20

μL of 5 mmol L-1 ferrozine. After 15 min, OD517 nm was measured. The inhibitory effect was

estimated according to equation (1), and EDTA at 100 μmol L-1 was used as the positive

control.

Ferric-reducing antioxidant power assay

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 This assay detects antioxidant activity by the reduction of ferric 2,4,6- tripyridyl-S-triazine

Fe(III)-TPTZ via the transformation to ferrous Fe(II)-TPTZ, detected by measuring OD700nm22.

In brief, 85 μL of 0.2 mol L-1 phosphate buffer (pH4.4) was mixed with 2.5 μL of 20% (w/v)

potassium, and then hexacyanoferrate(III) solution was added to 2.5 μL of each sample
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solution. After a 20-min water bath or dry bath at 50℃, the samples were ice bathed for 2

min. Subsequently, 160 μL of 10% (w/v) trichloroacetic acid was added to the sample and

was centrifuged at 3,000 × g for 10 min. After centrifugation, 75 μL of supernatant and 25 μL

of 0.2% (w/v) ferric chloride solution were added in 96-well. After 20 min, the absorption at

OD700nm was measured. BHA at 100 μmol L-1 was employed as the positive control.

Mushroom tyrosinase inhibition assay

Tyrosinase is the rate-limiting enzyme for managing the production of melanin23. Briefly, the

samples were first dissolved in aqueous DMSO and cultured in L-tyrosine (2.5 mg mL-1) with

phosphate buffer (pH6.8, 50 mmol L-1). All samples dissolved in DMSO, which is irrelevant to

tyrosinase activity examination when DMSO constitutes <0.5% (v/v) of the final working

volume. Subsequently, 25 U mL-1 of mushroom tyrosinase with an identical buffer was then

added (10 μL), and the solution was incubated at 37°C for 30 min. After 30 min, an ELISA

spectroscopic reader was applied to measure OD475nm, using 96-well microplates

(Molecular Devices, CA, USA). 2 mg of kojic acid dissolved in 20 μL (100 μmol L-1) was used

as a positive control. The inhibitory effect was obtained based on the following equation:

(2)

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 Whereas A is the OD 490 nm without the testing substance; B is the absorption without the

testing substance, but with tyrosinase; C is the absorption with the testing substance, and D

is the absorption with the testing substance but with tyrosinase.

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Collagenase inhibition assay

The assay used was based on spectrophotometry according our previously published

paper7. In the study, detection of collagenase activity inhibition is according to the protocol of

BioVision Collagenase Activity Colorimetric Assay Kit. Briefly, 1 µL of sample was mixed with

1 µL of collagenase (0.35 mU), 157 µL of collagen assay buffer and 40 µL of collagen. 160

µL of collagen assay buffer and 40 µL of collagen were used as the reaction substrate, as

the background vehicle control group. We measured the absorption at OD345 nm incubating

at 37℃ for 10 min interval. 2 µL of 1,10-phenanthroline (1.0 mol L-1) was used as positive

control. The inhibitory effect was obtained based on equation (2). Whereas A is the OD 345

nm without testing sample after incubation; B is the absorption without testing sample before

incubation; C is the absorption with sample after incubation; D is the absorption with sample

before incubation.

Cell culture and UV irradiation

Mouse melanoma B16-F10 cells and human fibroblast HS68 cells were obtained from the

Bioresource Collection and Research Center (BCRC number: 60031 and 60038, Hsinchu,

Taiwan). Two cells were cultured for 24 h in a 6-well plate with a cell density of 1 × 105 at 37

℃ in 5% (v/v) CO2 in DMEM supplemented with 10% (v/v) FBS, 1,000 IU mL-1 of penicillin,

0.1 mg mL-1 of streptomycin, and 0.25 μg mL-1 of amphotericin B22. The exposure dosage of

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 cells to UV irradiation (50 mJ cm-2, once) was based on our previous preliminary studies.

The medium was replaced with phosphate buffered saline (PBS) to wash it, and refill the

culture medium in turn for the treatments with HSCAE at various suitable concentrations for

24 h.
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Cell viability assay and pH adjustment

The effects of HSCAE on cell viability were estimated by the MTT method24. Cells were

cultured for 24 h in a 96-well plate with a density of 1  104, and then cultured for 4 h in a

serum-free medium. The medium was removed, and cells were incubated for another 24 h

with HSCAE at various concentrations from 0.05 to 10 mg mL-1, and the final volume was

100 µL. Subsequently, the medium was changed to a new medium with 0.5 mg mL-1 MTT

and incubated in 5% (v/v) CO2 at 37°C for two more hours. The medium was removed, and

the precipitate was dissolved in 100 µL DMSO to melt the formazan crystals, by shaking

lightly without light shine for 10 min, for maximum dissolution. The absorption at OD595nm was

detected to calculate the cell viability. The pH of all HSCAE experimental group samples

were measured by SevenEasy S20 pH meter (Mettler-Toledo Corp., OH, USA). First, the

instrument was calibrated with standard buffers of pH 4.0 and 7.0, and the electrode sensor

was cleaned with pure water between measurements to avoid errors. After the instrument

displayed a stable pH value, the HSCAE medium was diluted and adjusted to pH 7.3 for

various concentrations.

Cellular reactive oxygen species detection assay

To detect the intracellular ROS level, we used 2', 7’-Dichlorofluorescin diacetate (DCFDA,

Sigma-Aldrich Chemical Corp., D6883)25. 10 mmol L-1 DCFDA stock solution was prepared

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 with DMSO, followed by another dilution into a 20 µmol L-1 working solution with PBS. Cells

were incubated on a 12-well plate at the density of 1 × 105 and incubated for 1 day, followed

by an exposure to UVB and HSCAE for one more day. Harvested cells were blended with

the working solution for 30 min at 37°C, then analyzed via a flow cytometer (488 nm laser;
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Guava® easyCyte 5HT, Merck KGaA, Germany).

Collagen content assay

The concentration of collagen was determined using a commercially available ELISA kit

(Elabscience Biotechnology Corp., WuHan, China)7, 26. We collected cell culture supernatant

and centrifuged for 20 min with 1,000 × g. 100 μL of supernatant and collagen standards

were added to the 96-well plate, and the plates were incubated for 90 min at 37 °C. The

solution was removed, and 100 μL of the assay antibody working solution was added and

reacted for 1 h at 37°C. The solution was aspirated and washed at least three times. 100 μL

of horseradish peroxidase (HRP) conjugate working solution was added to each well and

incubated for 30 min at 37°C. After washing the well 5 times, 90 μL of the substrate solution

was added and incubated at 37°C for 15 min. To stop the reaction, 50 μL of stop solution was

mixed with each well. The commercial control sample was used as the blank vehicle group,

and absorbance value of each well at OD450nm was measured.

Total RNA isolation and extraction

Total RNA was extracted with Trizol RNA Isolation reagent (Sigma-Aldrich Chemical Corp.),

by isolating cell lysates and separating RNA, DNA and proteins. 1 mL of Trizol reagent was

added and incubated for 5 min. Subsequently, 200 μL BCP per mL Trizol were mixed in each

well and react for 2 min. The samples were centrifuged at 14,000 × g for 15 min, and the

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 supernatant was transferred into another Eppendorf tube. To precipitate RNA, isopropanol

was added in equal volume and mixed. The mixture was centrifuged at 13,000 × g for 15 min,

and the supernatant was removed. The sediment was washed with 1 mL of ethanol solution

75% (v/v) and centrifuged at 12,000 ×g for 5 min. Finally, the RNA pellet was dried and
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dissolved in the buffer solution treated with 50 µL diethyl pyrocarbonate (DEPC)27.

Detection of mRNA gene expression using qRT-PCR analysis

The qRT-PCR analysis was based on the method of AceQTM qPCR SYBR Green Master

Mix (High ROX Premixed; Agilent Corp., CA, USA) and our previous publication5. The

reaction solution was prepared to contain 10 µL 2  SYBR qPCR mix, 0.1 µL forward and

reverse primer, 1 µL RNA template, 2 µL of 50 × ROX reference dye, and deionized distilled

H2O to bring the total volume to 20 µL per well. The qPCR-PCR machine (ABITMStepOneTM

Plus, Thermo Fisher Corp., Waltham, MA, USA) was set up. First, the initial denaturation

temperature was set up at 95℃ for 15 min for one cycle. Second, the denaturation

temperature of 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s, and

the entire PCR stages were 40 cycles. The designed forward and reverse primers from 5′ to

3′ used in this experiment were shown in SI 1.

Western blotting analysis

A total of 1 × 105 B16-F10 or HS68 cells were treated with HSCAE groups and UVB

treatments or the blank vehicle control for one day, respectively28. The cells were harvested

and lysed with the lysis buffer (Thermo Scientific Pierce RIPA Buffer; 1 mmol L-1 EDTA, 10%

(v/v) glycerol, 1% (w/v) Nonidet P-40, 2 μmol L-1 leupeptin, 50 mmol L-1 Tris-HCl, 137 mmol

L-1 sodium chloride, 50 mmol L-1 sodium fluoride, 10 mmol L-1 sodium pyrophosphate, 20

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 mmol L-1 β-glycerophosphate, 1 mmol L-1 phenylmethylsulfonyl fluoride, 0.1 mmol L-1 sodium

orthovanadate and 2 μg mL-1 aprotinin; pH 7.5). Lysates were centrifuged at 13,000 × g for

30 minutes, and the protein quantification in the supernatant was assayed by bicinchoninic

acid (BCA) protein assay kit (Sigma-Aldrich Corp.) following centrifugation of lysates at
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12,000 rpm for 30 minutes. The amounts of protein were taken in equal quantities and

separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) on

12% (v/v) gel, and then electro-transferred to a polyvinylidene difluoride (PVDF)

nitrocellulose membrane (PALL Life Science, Ann Arbor, MI, USA). The transfer-film was

gently removed from the wet transfer tank, and semi-dry transfer slot, and then the PVDF

membrane was blocked with a buffer of 0.1% (v/v) Tween 20 for 1 h. After this, a mild rinse of

1 PBS-T was carried out to eliminate any traces of skim milk. In each case, the membrane

was incubated with a corresponding anti-mouse primary antibody to wash at least twice with

TBST buffer (PBS containing 0.1% (v/v) Tween 20) and dipped into horseradish

peroxidase-conjugated secondary antibodies against the corresponding primary antibody.

We used antibodies, including anti-β-actin (St John’s Laboratory, STJ97040), anti-MITF

(H-50) (Santa Cruz, sc-25386), anti-tyrosinase (H-109) (Santa Cruz, sc-15341), anti-TRP-1

(H-90) (Santa Cruz, sc-25534), anti-MMP1 (Biolegend, 634702), and anti-anti-TIMP1

(MA5-13688; Thermo Fisher Corp.). We added the descriptions about the multiple dilutions

of the various primary antibodies as following, Primary antibodies: anti-β-actin 1:5000,

anti-MITF 1:1000, anti-tyrosinase 1:1000, anti-TRP-1 1:1000, anti-MMP1 1:1000,

anti-TIMP1 1:200, respectively. This was then treated with enhanced chemiluminescence

(ECL) detection reagents (PerkinElmer, ECL1:ECL2=1:1) and exposed to A Mini Size

Chemiluminescent Imaging System from Life Science to specify the time intervals for

detecting the protein bands. Stained blots were visualized on a commercially available

imaging system.

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 Statistical Analysis

All experiments in each platform were performed in triplicate and expressed as the mean ±

standard error. For statistical analysis, all data were subjected to multiple comparative
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analysis by two-way ANOVA, followed by Dunnett’s post-hoc test as appropriate. Significant

differences (*) were defined as p < 0.05.

RESULTS

HSCAE had good antioxidant properties and free radical chelation ability

In this study, the antioxidant properties of HSCAE were assessed based on the DPPH

scavenging ability, reducing power and metal chelate activity. When the balance between

free radicals and intrinsic antioxidants is disturbed, ROS can produce lipid peroxide, DNA

damage, protein expression, tissue aging, and cancer occurrence. Free radical scavenging

properties are essential in functional nutrition for additives and skin care products. We dilute

HSCAE to target concentrations (20-100 mg mL-1). At the level of 60 mg mL-1 (62.78 ±

32.78%), the scavenging activity of free radicals scavenging ability exceeded vitamin C

(50.51 ± 1.41%) in Table 1. Also, the concentration of 100 mg mL-1 had the best free radical

scavenging activity, suggesting that free radical scavenging activity depended on HSCAE

concentration.

Iron is the most critical non-enzymatic catalyst for the initiation of lipid peroxidation, and

lipid peroxidation is the oxidative degradation of lipids. Fe (II) and FE (III) are the main

oxidizing states in biological systems and Fe (II) and ferrozine quantitatively form complexes.

Because of chelating agents, the formation of reagent complexes is often destroyed. We

dilute HSCAE to concentrations (20-100 mg mL-1). There was no significant chelating

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 capability at low levels (20-100 mg mL-1). However, at concentrations of 80 and 100 mg mL-1,

there were slight chelating capacities (15.69 ± 1.24% and 13.19 ± 1.63%), respectively. The

positive control EDTA had a chelating capacity of about 27% at 10 μmol L-1 in Table 1.

The last antioxidant assay is a ferric-reducing power test. In the experiment we

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measured the reduction capability and diluted HSCAE to concentrations (20-100 mg mL-1)

and BHA (10 μmol L-1) as the standard in Table 1. At low concentration of 40 mg mL-1,

HSCAE (absorbance: 0.81±0.01) had a better reduction capability than BHA (absorbance:

0.62 ± 0.05).

High concentrations of HSCAE were toxic to HS68 cells due to the low pH value

We then tested the appropriate concentration of HSCAE, and MTT assay was used to

determine the skin cell proliferation. We observed that HSCAE at low concentrations (0.1-2

mg mL-1) had no toxicity to HS68 and had a higher proliferation after treatment. HSCAE at

high concentrations (> 2 mg mL-1) had severe toxicity to HS68, and almost 80% of cells die

(Fig. 1A). In conclusion, the most suitable concentration of HSCAE used in cell experiments

was 0.1 – 2.0 mg mL-1. however, HSCAE showed cytotoxicity at high concentrations in cell

proliferation assays. We noted that HSCAE has a low pH value and its acidity may have

caused the cytotoxicity. The pH of the DMEM medium we used to incubate the cells was 7.3,

and the pH of the medium containing 5 mg mL-1 HSCAE was 6.89, which was significantly

toxic to the cells, and the pH of the medium with 10 mg mL-1 HSCAE was 6.56 in Fig. 1B. To

eliminate the effects of low pH, HSCAE was adjusted to pH 7 with sodium bicarbonate, and

cell proliferation was tested again. In Fig. 1B, the adjusted-pH HSCAE is less toxic to HS68

than the original. The original HSCAE at 5 mg mL-1 had 80% cytotoxic, but the toxicity of

adjusted-pH HSCAE was significantly reduced to 50%. We speculate that the significant

toxicity to cells may be due to the acidity, and the phenomenon of cell growth was recovered.

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 HSCAE reduced the UVB-induced ROS in HS68 cells

In vitro, HSCAE can eliminate DPPH free radicals; we confirmed that it could reduce

intracellular ROS production. The cells were treated in the same way as collagen
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quantitation assay then was analyzed by flow cytometry. The fluorescence intensity of the

highest peak in the control group was regarded as the standard value, and the peak shift of

the UVB group was observed. The ROS content of the UVB group was about 200% of the

control group, and the peak moved to the right, representing the increase of ROS intensity.

In the control group, the ratio to the left of the standard value was 41.8%, while the UVB

group was only 16%, which represented a rise in the proportion of UVB-induced HS68 cells

to produce high levels of ROS. The content of ROS in the UVB group treated with HSCAE

decreased significantly, and the peak shifted to the left with concentration dependence. At

0.1 mg mL-1, the ROS content was about 150% of the control group, the ratio was 22.2%, at

1 mg mL-1, the ROS content was about 140% of the control group, the ratio was 27.1%; at 2

mg mL-1, the ROS content was about 89% of the control group, and the ratio was 46.6%. We

confirmed that HSCAE could reduce the cellular ROS production with concentration

dependence in Fig. 2.

HSCAE achieved anti-aging effects by inhibiting collagenase activity and expression

The skin anti-aging effect was determined using qRT-PCR, Western blot, collagenase

activity inhibition, and collagen quantitation. This study examined the anti-aging ability of

HSCAE, proving its inhibition of collagenase activity, the enhancement of collagen content

and the reduction of collagenase expression. We measured the inhibition of collagenase

with suitable diluted HSCAE concentrations (1 - 100 mg mL-1). HSCAE showed no effect of

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 collagenase inhibition at low concentrations, but had a significant collagenase inhibition at

100 mg mL-1 (66.52 ± 14.47%) in Table 2. Next, mRNA expressions were quantified by

qRT-PCR. The UVB groups of HS68 cells were exposed to UVB (50 mJ cm-2). The UVB

group without HSCAE had higher mRNA levels of MMP-1 and MMP-3 that mainly degrade
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collagen in the ECM. The MMP-1 mRNA level in the UVB group was 6 folders compare to

the control group, while significantly reduced to 3 times of the control group after treated with

HSCAE at 1 mg mL-1. The MMP-3 mRNA level in the UVB group was two folders of control

group, while significantly lower than that in the control group after treated with HSCAE at 2

mg mL-1 (Fig. 3A). MMP and TIMP are antagonistic to each other that TIMP inhibited the

overexpression of MMP. When MMP expression was suppressed by HSCAE, it was

assumed that lower TIMP demand led to lower mRNA levels.

The protein expression of these genes was measured by Western blot, with β-actin as

the loading control. The MMP-1 expression level of UVB group was higher than that in the

control group and decreased after HSCAE treatment. The level of UVB group treated with

HSCAE at 0.1 mg mL-1 was 60% more than that of control, but only 10% at 2 mg mL-1. The

levels of UVB group and UVB group treated with HSCAE at 0.1 mg mL-1 were not elevated

but increased significantly when above 1 mg mL-1. At 1 mg mL-1, the level was 34% more

than the control group; at 2 mg mL-1, the level was 97% more than the control group in Fig.

3B. The contents of collagen in HS68 cell culture supernatant were quantified by ELISA

assay. After exposed to 50 mJ cm-2 UVB, cells were treated with HSCAE for one day. The

collagen content of the UVB group was 70% of the control group and increased by a dosage

dependence of HSCAE treatment. After HSCAE treatment at 2 mg mL-1, the collagen

content increased by 6% in Fig. 3C.

HSCAE achieved anti-melanin effects by inhibiting tyrosinase activity and expression

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 Aqueous H. Sabdariffa extracts have not yet been confirmed to have a whitening effect. This

study verified that HSCAE could reduce the activity of tyrosinase and expressions of MITF,

Tyrosinase, TRP-1, and TRP-2. We measured tyrosinase inhibitory effect from HSCAE at

various concentrations (1-100 mg mL-1). At low concentrations, HSCAE had no significant

Accepted Article

effect on the inhibition of tyrosinase activity; at 100 mg mL-1, 10% of tyrosinase activity was

suppressed in Table 2.

In addition to tyrosinase activity, mRNA expressions of melanogenesis were quantified

by using qRT-PCR. The whitening test used B16-F10 mouse melanoma cells, which were

treated in the same way as anti-aging tests, and UVB stimulated MITF and tyrosinase mRNA

expression levels. After treatment with HSCAE, the level of MITF and tyrosinase mRNA

decreased with concentration dependence. The MITF mRNA level in the UVB group was

three times that of the control group and decreased to two times after treatment with 2 mg

mL-1 HSCAE (Fig. 4A).

Protein expressions of the melanogenesis gene were measured by Western blot, and

β-actin was used as a loading control. The expression of MITF protein in the UVB group was

30% higher than that in the control group, and decreased after HSCAE treatment; at 1 mg

mL-1, the expression of UVB group was only 85% of the control group. The tyrosinase

expression in the UVB group did not increase significantly compared to the control group but

decreased after treating with HSCAE. At 2 mg mL-1, the expression of the UVB group was

only 52% of the control group. Changes of TRP-1 and TRP-2 were identical in the results of

qRT-PCR, so we chose TRP-1 to measure protein expression. TRP-1 and MITF protein

expression had the same trend; at 2 mg mL-1, the expression of TRP-1 was only 72% of the

control group (Fig. 4B). In summary, the results of qRT-PCR and Western blot echoed each

other; MITF, tyrosinase and TRP-1 protein were induced by UVB treatment, but inhibited by

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 HSCAE. HSCAE inhibits tyrosinase activity, reduces mRNA and protein expression, reduces

melanogenesis proteins, and has the potential for skin whitening.

Discussion
Accepted Article

HSCAE had good antioxidant resistance, but its low pH value was harmful to cells

In previous literature, it was found that the extract of H. sabdariffa has excellent antioxidant

activities. The different parts of H. sabdariffa were extracted in a variety of ways, leading to

various degree of antioxidant capability29. The seed and calyx had excellent antioxidative

power and radical-scavenging activity, and aqueous extracts had better scavenging activity

than ethanolic extracts; moreover, freeze-dried extracts showed higher scavenging activity30.

In addition, many beneficial foods and cosmetics also have antioxidant effects. To sum up, H.

sabdariffa as a common food has no obvious disadvantages to the human body. Aqueous

extract of H. sabdariffa calyx had good antioxidant and radical-scavenging activities, making

it suitable as a nutritious food ingredient and a potential cosmetic product component.

Before treating cells with HSCAE, it was essential to confirm whether HSCAE was

cytotoxic and to determine the appropriate concentration. We tried a serious of suitable

conditions to analogize UVB to induce skin aging. When cells were exposed to a dosage

over 100 mJ cm-2 UVB, severe cellular damage was observed. Based on preliminary

experiments, it the suitable experimental dosage is 50 mJ cm-2 UVB. Nutrients, osmotic

pressure, minerals, pH, etc., affect cell health; in addition to some essential elements

(including sodium, potassium, calcium, magnesium, nitrogen, and phosphorus), cells growth

requires trace elements, such as molybdenum, vanadium, iron, zinc and selenium, copper,

manganese, etc. Cell survival requires an optimal osmotic pressure at about 290 mOsm kg-1

in an isotonic environment. The cultured cells require an appropriate pH range, while most

cells have a pH range of 7.2 - 7.4; otherwise, it may have deleterious effects31. The low pH

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 value of HSCAE caused severe cytotoxicity, and the toxic effect of adjusted-pH HSCAE

(sodium bicarbonate buffer) was significantly reduced. Although toxicity was reduced, some

of the damage may still be due to other causes such as electrolytes, osmotic pressure, and

the like. In addition to the low pH value, we did not continue to explore the causes of
Accepted Article

cytotoxicity. Testing cosmetic products and the ingredients on animals was banned in the

United Kingdom in 1998 and across the European Union in 2013. The legislation is part of

European Union Regulation 1223/2009 (Cosmetics Regulation). In the United States, three

states have already passed laws that ban testing cosmetics on animals. Although the United

States Food and Drug Administration does not require animal safety testing for cosmetics —

a category which includes skin makeup, perfume, cream and shampoo — animal tests are

still used. Some countries, e.g., China, still require the tests. We try to avoid from animal

tests, but sometimes, it is a good examination for the safety and sensitivity tests. HSCAE is a

crude extract with some complex ingredients, and we thought it is better to do in vivo

experiments after confirming the cellular based in vitro characteristic results.

HSCAE could increase collagen and inhibit melanin-producing genes, but the effect

of aqueous extraction was limited

In Martino’s study, Hibiscus syriacus ethanolic extracts (HSEE) was applied to wound

healing, and HSEE stimulated collagen synthesis by 60% and promoted the wound healing32.

The oxidative stress on skin plays a major role in the aging process. Reactive oxygen

species (ROS) are dangerous oxygen molecules generated by UV rays and pollutions. In

human dermis, the disruption of the extracellular matrix (ECM) plays the most apparent role

which is true for intrinsic as well as extrinsic aging7. The results are fine wrinkles due to the

reduction of collagen, elastic fibers, and hyaluronic acid. We hypothesized that Hibiscus

plants had similar biological activity, and we confirmed that HSCAE protected collagen by

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 inhibiting the activity of collagenase. Although the results of Western blot showed that

adjustment of HSCAE to MMP-1 was less than anticipation, with the outcome of TIMP-1, we

could speculate that HSCAE had an anti-aging effect. For the change of TIMP-1 expression,

we had reached two conclusions. First, the increased expression of the TIMP-1 by HSCAE,
Accepted Article

and then TIMP-1 mRNA expressed might be reduced. Second, TIMP-1 increased

significantly and accompanied by a slight decrease in MMP-1, which cut collagen breakdown.

In general understanding, MMP-1 and TIMP-1 are antagonists that represent one rise with

another decline. Inflammatory cytokines, such as tumor necrosis factor-α, are reported to

increase the expression of MMPs and TIPM33. Inflammation occurs when the skin is

overexposed to UVB, and the antioxidant activity of HSCAE reduces inflammation and may

indirectly regulate the expression of MMPs and TIMP. In the study of Jha’s, it was also found

that MMP-1 and TIMP-1 decreased at the same time when the experimental autoimmune

anterior uveitis (EAAU) inflammation reaction decreased in late stage34. In our study, we

demonstrated that HSCAE could stimulate collagen synthesis, but the effect was much lower

than HSEE. We conjectured the reason for the disparity might be the difference between

plant varieties and extraction methods. Moreover, different cells used in the experiment also

had a significant impact on the results. However, we could inference the genus Hibiscus may

have some benefit to the skin.

In the study by Wong et al., several Hibiscus species were used to test anti-tyrosinase

activity35. The plants were powdered with liquid nitrogen and extracted using methanol, and

the methanol extract of H. sabdariffa leaves displayed antioxidants and tyrosinase inhibitory

ability, and total phenolic content was associated with the inhibition of tyrosinase activity.

Because H. sabdariffa has been reported to have a whitening effect, we speculate that

changing the extraction method can get better results. Tyrosinase is a critical enzyme to

produce melanin and oxidize tyrosine and dopamine to produce eumelanin and

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 pheomelanin; and some substances effectively whiten by inhibition tyrosinase activity, such

as kojic acid and arbutin36. In other words, the whitening effect of HSCAE could lead to its

cosmetics application for reducing melanin production. MITF is the transcription factor of

tyrosinase, TRP-1, TRP-2 and other protein associated with melanin production. We
Accepted Article

prospected that lower MITF mRNA level would coincide with TRP-1 and TRP-2 mRNA

reductions; however, these mRNA levels did not show the same trends. TRP-1 and TRP-2

mRNA levels of the UVB experimental group had no significant change, while were

increased slightly before declining, as like tyrosinase. Overall, HSCAE at 2 mg mL-1 had a

melanin inhibition capability by way of reducing mRNA expression. The process of melanin

production is an oxidation reaction; the use of antioxidants can interfere with the casein,

reducing the dopamine, interfering with pigment formation, and promote the metabolism of

melanin. HSCAE had excellent antioxidant properties, as well as melanin production

inhibitory activity.

HSCAE reduced the ROS produced by UVB in HS68 cells

The oxidative pressure can be generated by the external environment and internal

resources, causing some of the primary damage resulting from the peroxide (e.g., ROS,

superoxide radical, hydroxyl radical and hydrogen peroxide)37-44. The lack of antioxidants

leads to oxidative stress and excessive free radicals; peroxides cell membrane and even

mutates in deoxyribonucleic acid; affecting the structure of the protein and making the

function abnormal. ROS attack and react with stable skin cell molecules, causing

cross-linking of collagen and elastin while lessening skin’s ability to repair itself. We

emphasized the ability to decrease ROS production induced by an explosion to UVB and

antioxidant effectiveness of HSCAE. Matrix Metalloproteinases (MMPs) are enzymes

activated by UV exposure or inflammation. MMPs contribute to the breakdown of collagen

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 while inhibiting new collagen formation1. We confirmed that ROS induced by an explosion to

UVB was decreased, along with declining of MMP-1 and -3 expressions and enhancing of

TIMP-1 secretion. In another study, H. sabdariffa was also reported to reduce ROS. In the

study of Pattnaik et al., Saccharomyces cerevisiae cells were stimulated by H2O2 and
Accepted Article

treated with ethanolic extract of H. sabdariffa45. With the extract, the ROS content induced

by H2O2 reduced by more than 50%. HSCAE could achieve the same effect at 2 mg mL-1,

however HSCAE was prepared by aqueous extraction. All our other discussions had shown

that ethanolic extraction has good results in reducing ROS, our current result indicated that

the aqueous extract also has similar effects.

CONCLUSIONS

The experiments within this study showed that HSCAE ract repaired UVB-induced light

aging. The extract had an antioxidant capacity and inhibited intracellular ROS production in

a dose-dependent manner. We confirmed that HSCAE protected collagen from degradation

by inhibiting the expression and collagenase activity of MMP-1, and -9. As well as, HSCAE

inhibited the expressions of MITF, tyrosinase, and TRP-1, and tyrosinase activity to reduce

tyrosine metabolism into melanin. In conclusion, this study demonstrated that HSCAE had a

strong antioxidant capacity, the ability to maintain collagen production, melanogenesis

inhibition, and therefore it was as a potential skin anti-aging cosmetic ingredient.

AUTHORS’ CONTRIBUTIONS

Jian Li, Yi-Ru Lu, I-Fan Lin, Wenyi Kang, Hong-bin Chen, Hsu-Feng Lu and Hui-Min David

Wang conceived and designed the experiments; Yi-Ru Lu, Hsu-Feng Lu and I-Fan Lin

performed the experiments and analyzed the data; Hsu-Feng Lu, I-Fan Lin, Wenyi Kang,

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 Hong-bin Chen, Jian Li, and Hui-Min David Wang contributed the reagents, materials, and

analysis tools; Jian Li, Yi-Ru Lu, I-Fan Lin, Wenyi Kang, Hong-bin Chen, Hsu-Feng Lu and

Hui-Min David Wang wrote the paper.

Accepted Article

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Science and Technology (MOST

108-2221-E-005-044); we also thank the projects of Research Center for Sustainable

Energy and Nanotechnology, NCHU 107S0203B and project supported by the Natural

Science Foundation of Fujian Province, China (Grant No. 2017J01636).

CONFLICTS OF INTEREST

The authors have no competing interests regarding the publication of this study.

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 Tables

Table 1. The antioxidant effect of HSCAE

Concentration DPPH scavenging Metal chelating Reducing power

(mg mL-1) capacity (%) activity (%) (OD700)
Accepted Article

20 20.82 ± 9.56 ≦10.0 0.44 ± 0.01

40 37.92 ± 14.33 ≦10.0 0.81 ± 0.01

60 62.78 ± 32.78 ≦10.0 0.94 ± 0.06

80 70.86 ± 25.64 15.69 ± 1.24 1.26 ± 0.09

100 77.7 ± 19.57 20.96 ± 9.35 1.56 ± 0.10

Vitamin C a 95.14 ± 0.07 - -

EDTA b - 27.70 -

BHA c - - 1.50

(-) no testing
a. Vitamin C (10 mmol L-1) was used as a positive control for DPPH assay.
b. EDTA (10 mmol L-1) was used as a positive control for chelating assay.
c. BHA (10 mmol L-1) was used as a positive control for reducing power assay.

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 Table 2. The anti-tyrosinase and anti-collagenase effects of HSCAE

Concentration Mushroom tyrosinase

Collagenase inhibition (%)
(mg mL-1) inhibition (%)

1 ≦10.0 14.59 ± 1.22

Accepted Article

5 ≦10.0 9.23 ± 0.77

10 ≦10.0 8.16 ± 0.01

50 25.88 ± 3.13 9.39 ± 1.53

100 66.52 ± 14.47 11.84 ± 0.01

Kojic acid a - 76.63 ± 0.14

1,10-Phenanthroline b ≧90.0 -

(-) no testing
a. Kojic acid (0.1 μg μL-1) was used as a positive control for mushroom tyrosinase assay.

b. 1,10-Phenanthroline (1 mol L-1) was used as a positive control for collagenase inhibition

assay.

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 FIGURE CAPTIONS
Figure No. Caption

(A) HS68 cells in a 96-well microtiter plate at approximate densities of 1  104

cells per well. After treatment with 0.1, 0.5, 1, 2, 5 and 10 mg mL-1 of HSCAE for
Accepted Article

24 h, the viabilities of HS68 cells were measured by MTT assay. (B) HS68 cells
Fig. 1 treated with adjusted-pH HSCAE, the viabilities were measured. All data were

subjected to multiple comparative analysis by two-way ANOVA, followed by

Dunnett’s post-hoc test as appropriate. Significant differences (*) were defined

as p < 0.05; (**) were defined as p < 0.001.

The DCFDA assay results showing that HSCAE treatment decreased ROS

production in HS68 cells. All data were subjected to multiple comparative

Fig. 2 analysis by two-way ANOVA, followed by Dunnett’s post-hoc test as

appropriate. Significant differences (*) were defined as p < 0.05; (**) were

defined as p < 0.001.

The qRT-PCR and Western blot assay results showing that HSCAE treatment

decreased MMP-1 and MMP-3 expression and increased TIMP-1 expression in

HS68 cells. ELISA assay results showing that HSCAE treatment increased
Fig. 3 collagen content. All data were subjected to multiple comparative analysis by

two-way ANOVA, followed by Dunnett’s post-hoc test as appropriate.

Significant differences (*) were defined as p < 0.05; (**) were defined as p <

0.001.

The qRT-PCR and Western blot assay results showing that HSCAE treatment

decreased MITF, tyrosinase and TRP-1 expression in HS68 cells. All data
Fig. 4
were subjected to multiple comparative analysis by two-way ANOVA, followed

by Dunnett’s post-hoc test as appropriate. Significant differences (*) were

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 defined as p < 0.05; (**) were defined as p < 0.001.

Fig. 5 Graphical abstract

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 Fig. 1
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 Fig. 2
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 Fig. 3
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 Fig. 4
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 Fig. 5
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