Pharmacological Research - Modern Chinese Medicine 5 (2022) 100202

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Pharmacological Research - Modern Chinese Medicine

journal homepage: www.elsevier.com/locate/prmcm

Evaluation of the extract of traditional Chinese medicine formula Si Ben

Cao for skin whitening
Yu-Shan Lin a, Hong-Yu Peng a, Yu-Xin Zhu a, Yu-Xiao Meng a, Hui-Hui Xiao a,b,c,∗,
Guo-Qing Chen a,b,c,∗
a
State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), Shenzhen Research Institute, The Hong Kong Polytechnic University, Shenzhen
518057, Hong Kong Special Administrative Region
b
Research Centre for Chinese Medicine Innovation, The Hong Kong Polytechnic University, Hung Hom, Hong Kong Special Administrative Region
c
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong Special Administrative Region

a r t i c l e i n f o a b s t r a c t

Keywords: Objective: To evaluate the safety, potential benefits and possible mechanisms of the extract of Si Ben Cao (SBC),
Skin whitening a traditional Chinese medicine formula composed of four herbs, for skin whitening.
Traditional Chinese medicine formula Methods: The reflux method was performed to prepare the extract of SBC. MTT assay was used to evaluate the
Tyrosinase inhibitor
cytotoxic of SBC extract in vitro. Skin irritation test and skin allergy test on rats were performed to evaluate the
Melanin reduction
safety of SBC extract in vivo. Intercellular melanin production was analyzed to assess the function of SBC extract
for whitening. Afterwards, we analyzed the activity of intracellular tyrosinase, Ultraviolet (UV) absorption, and
anti-oxidant activity, which were related to melanin production.
Results: The aqueous extract and ethanol extract of SBC were prepared separately. MTT assay results showed
aqueous extract of SBC at a concentration of 100 𝜇g/mL had no inhibitory effect on the proliferation of B16F10
cells even after treatment for 96 h, indicating that aqueous extract of SBC was not cytotoxic. Thus, we conducted
subsequent experiments using aqueous extract of SBC. The results on rats showed SBC extract was safe to apply on
the skin without causing irritation and allergies. Analysis of melanin content showed SBC extract had inhibitory
effect on cellular melanin generation and secretion. Moreover, we found that SBC extract was able to reduce
cellular tyrosinase activity, as well as absorb UV light and scavenge DPPH radicals for anti-oxidants.
Conclusion: Our data clearly demonstrated that SBC extract is safe and effective for skincare, and it is suitable
for use with the purpose of skin whitening and health benefits.

1. Introduction including melisma, phelides, and dark spots on the skin [5]. Melano-
genesis is the process of melanin synthesis, which is produced in the
Skin is the largest organ in the human body, weighing 3.6 kg and cov- melanosomes of melanocytes [6]. During melanogenesis, tyrosinase,
ering an area of 2 m2 . It is the first defense line of the body, protecting the enzyme mainly responsible for melanin production, catalyzes the
us against extreme temperatures, and external physical and biological hydroxylation of L-tyrosine to L-DOPA (L‐3,4‐dihydroxyphenylalanine)
invasions [1]. In recent years, people have been paying more attention and the oxidation of L-DOPA to DOPA quinone [7]. Due to this, inhi-
to the health of their skin due to improving living conditions. In Asian bition of tyrosinase has been regarded as a long-term method of pre-
countries, fair skin has always been associated with youth, beauty, and venting hyperpigmentation [8,9]. In response, cosmetic companies and
health, especially for young women [2]. There is a saying in China that researchers are engaged in developing new whitening agents that inhibit
one white hides a hundred ugly things. Thus, a variety of cosmetic skin tyrosinase activity [10].
whitening products are developed to satisfy the desire for fair skin [3]. Recently, scientists have been exploring natural sources like herbs
Skin contains melanin, which is a natural dark pigment produced for tyrosinase inhibitors [8,11]. However, it has proven difficult to find
by melanocytes [4]. Normally, melanin protects the skin from Ultravi- compounds that are highly effective, safe, and have low side effects. In
olet (UV) damage, but excessive production of melanin leads to hyper- China, traditional herbs have been safely used to treat diseases, includ-
pigmentation, which can contribute to a variety of aesthetic problems, ing skin whitening and skincare, for many years [12,13]. Generally, a

∗
Corresponding authors at: State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), Shenzhen Research Institute, The Hong Kong
Polytechnic University, Shenzhen, Guangdong 518057, Hong Kong Special Administrative Region
E-mail addresses: huihui.xiao@polyu.edu.hk (H.-H. Xiao), guoqing.chen@polyu.edu.hk (G.-Q. Chen).

https://doi.org/10.1016/j.prmcm.2022.100202
Received 9 November 2022; Received in revised form 28 November 2022; Accepted 29 November 2022
Available online 30 November 2022
2667-1425/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Y.-S. Lin, H.-Y. Peng, Y.-X. Zhu et al. Pharmacological Research - Modern Chinese Medicine 5 (2022) 100202

combination of several herbs is prescribed for use, based on the unique 𝛼-MSH (100 nM), SBC and four herbs extracts were added at the con-
theory of traditional Chinese medicine (TCM), referred to as TCM for- centration of 100 𝜇g/mL, separately. When the treatment time achieved
mula [14]. A formula containing several herbs has more efficacy and low at 96 h, cells were ruptured by freezing and thawing. Then cell lysates
toxicity than a single herb or compound, which is currently attracting were collected by centrifugation. After protein concentrations of lysates
the attention of the cosmetic and medical industries [15]. were determinate by PierceTM BCA Protein Assay (Thermo Scientific),
In here, we report on a TCM formula called Si Ben Cao (SBC), made the concentrations of all lysates were adjusted to be the same. Then, cell
up of four Chinese traditional herbs. The aqueous extract of SBC is ef- lysates were mixed with L-DOPA (0.1% in PBS), incubated at 37 °C for
fective in inhibiting tyrosinase activity, decreasing melanin generation, 30 min, and the absorbance was measured at 475 nm. The tyrosinase
and absorption of UV rays, which is much better than herbs alone. Fur- activity was represented as percentage of the vehicle.
thermore, it has no irritation and allergy to skin.
2.6. Western blot analysis
2. Materials and methods
B16F10 cells were seeded at 3 × 104 cells per well in a 6-well plate
2.1. Preparation of SBC extract and were stimulated with 𝛼-MSH (100 nM). After 24 h, cells were ex-
posed to extracts at the concentration of 100 𝜇g/mL for 96 h. Then whole
All four herbs of SBC were purchased from the PuraPharm Corpo- cell lysates were collected and suspended in lysis buffers according to
ration. In order to apply for a patent for the combination of four herbs the report [22]. Following centrifugation at 13,500 rpm for 15 min at
(SBC formula), we choose not to disclose the names of the herbs, which 4 °C, total protein concentration was measured by PierceTM BCA Pro-
are represented by A, B, C and D. The four herbs were authenticated tein Assay (Thermo Scientific), and 10 to 25 𝜇g of protein was separated
by Dr. Sibao Chen based on morphological features. In order to prepare on 10% SDS-PAGE and transferred to PVDF membranes. After blocking
aqueous extracts, all the herbs were soaked in distilled water for 1 h, sep- (5% skim milk powder in TBST) for 1 h at room temperature, the mem-
arately. Then the extract was collected using the reflux method for 2 h brane was then incubated with tyrosinase antibody (Beyotime, AF8283)
according to the method in the publication [16]. After being lyophilized overnight at 4 °C. The membrane was incubated with secondary anti-
into powder, we got the aqueous extracts of A, B, C, D and SBC respec- body for 1 h at room temperature. All antibodies were diluted in TBST
tively. For preparing ethanol extracts, ethanol replaced water and same containing 5% dry milk. The immune-reactive proteins were detected by
protocol was performed. enhanced chemiluminescence (ECL) using X-ray film and ECL reagent.

2.2. Cell culture 2.7. DPPH radical scavenging activity

B16F10 cells were purchased from American Type Culture Collec- In accordance with the report [23], DPPH was used for assessing
tion (Manassas, USA). Cells were cultured in DMEM supplemented with the free radical scavenging activity. The various concentration of herb
10% FBS in a humidified atmosphere containing 5% CO2 and 95% air extracts at 20 𝜇L were added to 180 𝜇L of 250 𝜇M DPPH solution. The
at 37 °C. The medium was changed every three days, and cells were reaction was incubated at 25 °C for 30 min in the dark. Then, optical
passaged using 0.05% trypsin/EDTA. density (O.D.) values were measured at 517 nm on a microplate reader,
and the O.D. for untreated group were set as 100% viability.
2.3. MTT assay
2.8. UV absorption assay
The effect of SBC extract on proliferation and viabilities of
B16F10 cells was determinated by the 3-(4,5-dimethylthiazol-2-yl)-2,5- UV absorption assay was performed according to the publication
diphenyltetrazolium bromide (MTT) uptake method according to the re- [24]. SBC and four herbs extracts were dissolved in aqueous solutions,
port [17]. In generally, 5 × 103 cells were seed in each well of a 96-well then UV absorption spectra was analyzed on a UV spectrophotometer at
plate. After 24 h, cells were exposed to extracts at the concentration of room temperature using a quartz cuvette as the holder.
100 𝜇g/mL for 24, 48, 72 and 96 h. Kojic acid (KA) was used as a posi-
tive control and it was added into cells at the concentration of 100 𝜇M 2.9. Animal study
[18]. The optical density (O.D.) values for untreated group were set as
100% viability. The animal welfare and all experimental protocols were approved by
the Animal Ethic Committee of The Hong Kong Polytechnic University.
2.4. Measurement of melanin production A total of 35 Specific-Pathogen-Free (SPF) Sprague-Dawley (SD) rats
(female 20, male 15, four-month old) were purchased from Beijing Vital
B16F10 cells were seeded at 1.5 × 104 cells per well in a 12-well River Laboratory Animal Technology Co., Ltd. The rats were randomly
plate and stimulated with 𝛼-MSH (100 nM) [19], while SBC and four divided to 5 group (4 females and 3 males for each group). During the
herbs extracts were added at the concentration of 100 𝜇g/mL, sepa- study, all rats were housed in the standard condition of 23 ± 2 °C, 40%–
rately. When the treatment time achieved at 96 h, melanin production 60% humidity and an alternating 12 h light/12 h dark cycle, and all rats
was measured according to the report [20]. The culture mediums treated were allowed free access to normal diet and distilled water.
with different extracts were directly measured at 475 nm to determinate
the content of extracellular melanin secretion. For intracellular melanin 2.10. Skin irritation test
production, cells were washed with PBS and lysed with NaOH (1 M)
containing 10% DMSO for 2 h at 80 °C. After that, cell lysates were The skin irritation test was performed as previously reported
centrifuged and supernatants were measured at 475 nm. The melanin [25] and included intact skin group and scratched skin group (female
content was represented as percentage of the vehicle. 4 and male 3 for each group). Two sides on the backs of all rats were
depilated (3 cm2 /each) using hair removal cream (Veet, French). A day
2.5. Intracellular tyrosinase activity assay later, both sides of the rats in scratched skin group were scratched us-
ing a sandpaper, causing the skin slightly ooze blood. Then the vehicle
Intracellular tyrosinase activity was measured according to a method and SBC extract was applied daily on left side and right side in both
in the publication [21]. Following the seeding of B16F10 cells at a den- groups, respectively, for 7 days. At the last application day, the sub-
sity of 1.5 × 104 cells per well in a 12-well plate and stimulating with stances were removed by water after 1 h treatment, then erythema and

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Y.-S. Lin, H.-Y. Peng, Y.-X. Zhu et al. Pharmacological Research - Modern Chinese Medicine 5 (2022) 100202

edema scoring was performed at 1 h, 24 h, 48 h, 72 h and 7 days. A

zero-four scale was used to determine the erythema and edema as fol-
lows: zero, no erythema/edema; one, very slight (barely perceptible);
two, well defined; three, moderate to severe; and four, severe (beet red-
ness erythema to mild eschar formation/edema raised more than 1 mm
and extending beyond the exposure area). The Irritation Index (I.I.) was
calculated according to the equation: I.I. = (erythema grades at all-time
points + edema grades at all-time points)/ (number of rats).

2.11. Skin allergy test

The skin allergy test was performed as previously reported [26] and
rats were randomly divided into 3 groups, including 4 females and 3
males for each group. As the irritation test, all rats were depilated 24 h
before the test. Then, the right side of rats was applied dermally with
vehicle, positive control (2,4-Dinitrochlorobenzene, 1% in acetone) and
SBC extract, respectively, on days 1, 7 and 14. The substances were
allowed to stay on the skin for 6 h, then they were removed by water.
On day 28, the left side of the rats was treatment once with the same
method. After the last treatment, the erythema and edema scoring was
performed at 6 h, 24 h, 48 h and 72 h. The erythema was graded by
a zero-four scale as follows: zero, no erythema; one, very slight; two,
well defined; three, moderate to severe; and four, severe (edematous
erythema), while the edema was graded to a zero-three scale as follows:
zero, no edema; one, very slight (barely perceptible); two, well defined;
three, severe (raised more than 1 mm with clear contours). The allergy
Index (A.I.) was calculated as same as I.I. The allergy rate (A.R.) was
calculated by the equation: A.R.= (number of allergy rats/ number of
total rats) × 100%.

2.12. Statistical analysis

Each experiment was performed at least three times. GraphPad Prism

5.0 software was used for statistical analysis.

3. Results

3.1. Aqueous extract of SBC has no cytotoxicity

Firstly, we separately prepared ethanol and aqueous extracts for SBC

formula and individual herbs. To determine the cytotoxicity of extracts,
MTT assay was performed to analyze the viability of B16F10 melanoma
cells under treatment of extracts. Kojic acid (KA), a skin-lightening Fig. 1. Cell viabilities were measured by MTT assay at four time points 1. Con-
trol; 2. KA; 3-6. ethanol extracts of individual four herbs (A, B, C and D); 7.
agent, was used as a positive control at the concentration of 100 𝜇M
Ethanol extract of SBC; 8-11. aqueous extracts of individual four herbs (A, B,
[27]. Briefly, cells were cultured in a 96-well plate and treated with dif-
C and D); 12. aqueous extract of SBC; ∗ P < 0.05, ∗ ∗ P < 0.01, compared with
ferent extracts at the concentration of 100 𝜇g/mL for 24 h, 48 h, 72 h control group.
and 96 h. The results in Fig. 1 were showed as relative cell viability com-
pared with control. We found that KA significantly inhibited the prolif-
eration of B16F10 cells at the first time point (24 h), and its inhibitory conducted subsequent experiments using aqueous extract of SBC, and
activity was enhanced with time extended. As well as KA, the ethanol all extracts mentioned later were aqueous extracts.
extract of A, B, C, D and SBC also showed inhibitory effects. Obviously,
the inhibitory effect of SBC ethanol extract was stronger than the ethanol 3.2. SBC extract has no irritation and allergy to skin
extracts of the four herbs individually. Intriguingly, except for aqueous
extract of D showing inhibitory activity in 72 h and 96 h, the aqueous The results of irritation index and allergy index of SBC extract on rats
extracts of A, B, C and SBC had no inhibitory effect. These results in- were shown in Table 1 and Table 2, respectively, while the images of
dicated that aqueous extract of SBC was non-cytotoxic. We therefore them were exhibited in Fig. 2. As shown in Table 1, there were slight ir-

Table 1
Values of the irritation index determined in SD rats (female 4 and male 3/each group).

Group Treatment n average score Irritation index

1h 24h 48h 72h 7d

Intact skin Vehicle 7 0.43 0.36 0 0 0 0.79

SBC extract 7 0.14 0.07 0 0 0 0.21
Scratched Vehicle 7 0.14 0 0 0 0 0.14
skin SBC extract 7 0.14 0 0 0 0 0.14

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Y.-S. Lin, H.-Y. Peng, Y.-X. Zhu et al. Pharmacological Research - Modern Chinese Medicine 5 (2022) 100202

Table 2
Values of the allergy index determined in SD rats (female 4 and male 3/each group).

Group n Number of allergy rats 6h 24h 48h 72h Allergy index Allergy rate (%)

Vehicle 7 0 0 0 0 0 0 0
Positive control 7 7 2 1 0.57 0.29 3.86 100
SBC extract 7 0 0 0 0 0 0 0

Fig. 2. Images of skin irritation and allergy on SD rats A: image of skin irritation.
The vehicle and SBC extract was applied daily on 4-month old SD rats (female
4 and male 3 for each group) after scratching or not for 7days; B: image of skin
allergy. The SD rats (4-month old, female 4 and male 3 for each group) was
applied dermally with vehicle, positive control (2,4-Dinitrochlorobenzene, 1%
in acetone) and SBC extract, respectively, on days 1, 7 and 14.

ritations, no matter in intact skin and scratched skin group treated with
or without SBC at 1 h, while the irritation disappeared over time, which Fig. 3. Inhibition of melanin synthesis by SBC extract A. Melanin content in
suggesting that the irritations might induced by hair removal cream. In culture medium was measured after treatment for 96 h, ∗ ∗ P < 0.01; B. Melanin
the right side, SBC treatment did not cause any irritation in scratched content in cells was measured after after treatment for 96 h, ∗ ∗ P < 0.01; C.
skin rats compared with intact skin rats. The allergy test showed that the Photo of cell pellets after treatment for 96 h.
positive control of 2,4-Dinitrochlorobenzene showed an obvious mod-
erate allergenicity compared with vehicle group, while SBC extract did
not induce any allergy at all-time points (Table 2). The above results ited the increase of melanin in cells and in culture medium. Compared
demonstrated that the SBC extract was neither irritating or allergenic with KA, SBC extract resulted in more drop of melanin content, both
to skin, suggesting SBC extract was safe enough for application on the in cells and in culture medium. The results indicated that SBC extract
skin. has inhibitory effect on melanin generation and secretion. Interestingly,
SBC extract had a greater inhibitory effect than the four extracts taken
3.3. SBC extract decreases melanin content alone, although they both decreased melanin content. As seen visually in
Fig. 3C, cells became extremely black under 𝛼-MSH stimulation, while
Next, we examined melanin formation regulated by SBC extract in SBC extract-treated cells appear the whitest, except for control cells,
B16F10 cells and culture mediums. 𝛼-MSH (100 nM) was added to the which did not receive 𝛼-MSH stimulation.
plates after cells were seeded, followed by the separate addition of ex-
tracts from SBC and four individual herbs at the concentration of 100 3.4. SBC extract inhibits tyrosinase activity
𝜇g/mL. Melanin content was measured after 96 h of treatment, and
it was represented as a percentage of control. Results in Fig. 3A and SBC extract was assessed for its inhibitory effect on tyrosinase ac-
3B showed melanin content in cells and in culture medium increased tivity in B16F10 melanoma cells. In accordance with the protocol in
sharply following 𝛼-MSH stimulation. As a positive control, KA inhib- analysis of melanin content, cells were stimulated with 𝛼-MSH, fol-

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Y.-S. Lin, H.-Y. Peng, Y.-X. Zhu et al. Pharmacological Research - Modern Chinese Medicine 5 (2022) 100202

obvious ability to absorb UV ray, especially between 200 and 290 nm.
With the concentration of SBC extract increased from 62.5 𝜇g/mL to 500
𝜇g/mL, its intensity of UV absorption was also significantly increased
(Fig. 5A), confirming that SBC extract had the activity of UV absorp-
tion. Subsequently, UV absorption of SBC extract and the four herbs
extracts alone were compared as shown in Fig. 5B. There was moderate
UV absorption by C and D, but relatively poor UV absorption by A and
B. Interestingly, with these four herbs combined into SBC formula, the
overall intensity was greatly enhanced. In particular, although only C
produced a peak in UV absorption at 250-290 nm, the intensity of this
peak regulated by the SBC remained much stronger than that regulated
by C alone.

3.6. SBC extract shows anti-oxidant activity

UV ray can cause free radicals generation in human skin [31]. As a

result of protectable mechanism, melanin production is strongly corre-
lated with free radicals [32]. 𝛼, 𝛼-diphenyl-𝛽-picrylhydrazyl (DPPH), a
free radical, is usually be used for the colorimetric evaluation of anti-
Fig. 4. Inhibition of tyrosinase activity by SBC extract A. Intracellular tyrosinase oxidant radical scavenging activity [33]. In this study, we measured the
activity was measured after treatment for 96 h, ∗ ∗ P < 0.01; B. The expression anti-oxidant activity of the SBC extract using the DPPH assay. Accord-
of tyrosinase was analyzed by western blot after treatment for 96 h. ing to Fig. 6A, when the concentration of SBC extract was 50 𝜇g/mL,
the free radical scavenging rate was up to 49.65 ± 0.57%. Following
the concentration increased, the free radical scavenging rate was also
lowed by SBC extract and extracts of four herbs at the concentration increased, confirming that SBC extract had anti-oxidant activity with a
of 100 𝜇g/mL. KA was also used as a positive control. The activity dose-dependent manner. In addition, anti-oxidant activities of SBC and
of cellular tyrosinase was measured after 96 h of treatment, and it the four herbs at the same concentration of 100 𝜇g/mL were compared
was represented as a percentage of the control group [28]. Results in in Fig. 6B. Consistently, SBC extract also showed much more effective
Fig. 4A showed that 𝛼-MSH greatly increased intracellular tyrosinase at scavenging DPPH radicals than the four herbs alone.
activity to 698.64 ± 21.43%. As a positive control, KA decreased it to
329.25 ± 9.64%. There was a degree of inhibitory activity observed 4. Discussion
in extracts from four herbs. Interestingly, SBC, as a formula that com-
bined these four herbs, showed stronger effect than KA and four extracts In modern society, cosmetics are used to change people’s appearance
alone, which was 213.60 ± 13.12%. Additionally, western blot assay was to enhance their beauty and attractiveness, which has been considered
used to analyze the expression of tyrosinase protein. Results in Fig. 4B an indispensable commodity. Skin whitening products are a major cat-
showed that 𝛼-MSH stimulation significantly increased the expression egory of cosmetics in the world, especially in eastern Asian countries.
of this protein, but KA could not affect this increase. As expected, SBC In recent years, skin whitening products containing natural ingredients
extract also inhibited the expression of this protein, confirming that SBC from herbs have gained increasing attention [34,35]. Since TCM formu-
extract inhibited tyrosinase activity. las have long been safely used to improve complexions, developing skin
whitening products based on them is more feasible. Therefore, we eval-
3.5. SBC extract shows Ultraviolet absorption uated the potential of our SBC formula as a skin whitening agent in light
of scientific research.
Ultraviolet (UV) ray is common in life, it can activate melanin syn- Because whitening agents are used on the skin, it is the most im-
thesis in skin [29]. Therefore, a reagent with skin whitening function portant that agents need to be safe, non-toxic, and non-irritating to the
is best to absorb UV. The absorption of UV radiation at wavelengths skin [36]. In our study, to evaluate the safety of SBC extract for skin ap-
of 200–400 nm was analyzed [30], and we found SBC extract had an plication, MTT assay was performed that examines the inhibitory effect

Fig. 5. SBC extract absorbs UV ray A. the UV

absorb curve of SBC extract with concentra-
tions from 62.5 𝜇g/mL to 500 𝜇g/mL; B. the UV
absorb curve between SBC extract and extracts
of individual four herbs with concentration of
500 𝜇g/mL.

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Y.-S. Lin, H.-Y. Peng, Y.-X. Zhu et al. Pharmacological Research - Modern Chinese Medicine 5 (2022) 100202

Fig. 6. The OD value was measured in DPPH

radical scavenging assay A. The OD value reg-
ulated by SBC with the concentrations from 50
𝜇g/mL to 200 𝜇g/mL, ∗ ∗ P < 0.01; B. The OD
value regulated by SBC extract and extracts of
individual four herbs at the concentration of
100 𝜇g/mL, ∗ ∗ P < 0.01.

of on B16F10 cells proliferation. We found KA, a skin whitening ingre- therapies, like TCM formulas, have better effectiveness and fewer side
dient that typically has a limited concentration, and ethanol extract of effects than single drugs [45]. Our study revealed that SBC formula has
SBC to have moderate inhibitory activity on the proliferation of B16F10 several advantages over single herbs in preventing hyperpigmentation,
cells. In contrast, aqueous extract of SBC had no effect on B16F10 cells and it is not toxic after combining, which is a TCM advantage.
proliferation, even after 96 h of incubation, confirming that aqueous
extract of SBC is non-cytotoxic. Thus, instead of ethanol extract of SBC, 5. Limitations
aqueous extract of SBC was used in our subsequent experiments. It is in-
teresting that aqueous extraction is accordance with TCM practices for This study has some limitations. Due to medical ethics and our lim-
using herbs. Furthermore, skin irritation and skin allergy were analyzed ited financial sources, we were unable to test the whitening effect of
on rats. The results of non-irritating and non-allergic on rats confirmed SBC extract directly on the human body. Additionally, functions of SBC
that of non-cytotoxicity, which all suggested that SBC extract is safe for extract may be different from its cosmetic products, because cosmetic
skin application. products usually contain a variety of substrates and added functional
Melanin is an important component of skin, and its quantity deter- ingredients. Further work should address these issues.
mines the color of skin [6]. In this study, we examined the content of
intercellular and extracellular melanin regulated by SBC extract. Com- 6. Conclusions
pared with KA, SBC extract showed significantly greater efficacy in re-
ducing both intracellular and extracellular melanin levels. Melanin is In summary, our findings indicate that aqueous extract of SBC, a TCM
synthesized by tyrosinase, an oxidoreductase widely present in skin. For formula, is safe to apply on the skin with no irritation and allergy. And it
the development of skin whitening products, tyrosinase inhibition is re- not only inhibits tyrosinase and melanin production, but also scavenges
garded as one of the most effective strategies [37]. According to our free radicals and absorbs UV rays. It is suggested that a cosmetic product
study, SBC extract significantly reduced the activity of cellular tyrosi- containing SBC extract could be developed for skin whitening and health
nase compared to KA. It is known that KA is a tyrosinase inhibitor, but benefits.
it does not decrease tyrosinase expression in several B16 cell lines [38].
In contrast to KA, SBC extract showed a strong inhibitory effect on tyrosi- Formatting of funding sources
nase expression, suggesting SBC extract may have different mechanisms
for inhibiting tyrosinase activity. The study was supported by The Hong Kong Polytechnic University
Additionally, melanin synthesis in the skin is also stimulated by ex- Start-up Fund (P0038607 and P0038596), and 100 Cities and 100 Insti-
ternal factors such as ultraviolet (UV) rays [39] and free radicals [40]. tutes Special Launch Fund (P0039502).
The UV rays can generate free radicals, and free radicals are involved
in the reaction of tyrosinase, which leads to melanin production [41]. Declaration of Competing Interest
More importantly, UV rays can cause more damage to people’s skin if
exposed for a long period of time. It is therefore better for cosmetic The authors declare no conflict of interest.
products that have whitening properties to also have UV filter and anti-
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