Mechanisms of white mustard seed (Sinapis alba L.) volatile oils as transdermal penetration enhancers

Accepted Manuscript Mechanisms of white mustard seed (Sinapis alba L.) volatile oils as transdermal penetration enhancers

ShiFa Ruan, ZhuXian Wang, ShiJian Xiang, HuoJi Chen, Qun Shen, Li Liu, WenFeng Wu, SiWei Cao, ZongWei Wang, ZhiJun Yang, LiDong Weng, HongXia Zhu, Qiang Liu PII: DOI: Article Number: Reference:

S0367-326X(19)30482-4 https://doi.org/10.1016/j.fitote.2019.104195 104195 FITOTE 104195

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

5 March 2019 2 June 2019 4 June 2019

Please cite this article as: S. Ruan, Z. Wang, S. Xiang, et al., Mechanisms of white mustard seed (Sinapis alba L.) volatile oils as transdermal penetration enhancers, Fitoterapia, https://doi.org/10.1016/j.fitote.2019.104195

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ACCEPTED MANUSCRIPT Mechanisms of white mustard seed (Sinapis alba L.) volatile oils as transdermal penetration enhancers ShiFa Ruana1, [email protected] , ZhuXian Wanga,1, [email protected]. , ShiJian Xianga, HuoJi Chena, Qun Shena, Li Liua, WenFeng Wua, SiWei Caoa, Wangb,

ZhiJun

Yangc,

LiDong

Wenga,

: School of Traditional Chinese Medicine, Southern Medical University, Guangzhou

SC

a

Zhud*,

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[email protected], Qiang Liua*, [email protected]

HongXia

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ZongWei

510515, China

: Beth Israel Deaconess Medical Center, Urologic Surgery Section, Harvard Medical

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b

: School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China

d

: Integrated Hospital of Traditional Chinese Medicine, Southern Medical University,

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Guangzhou, 510300, China

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c

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School,330 Brookline Ave, Boston, MA 02215 , USA

*Corresponding author.

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ABSTRACT

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We investigated the transdermal drug permeation enhancement properties and associated mechanisms of white mustard (Sinapis alba L.) seed volatile oil (SVO).

1

ShiFa Ruan and ZhuXian Wang contributed equally to this work.

ATR-FTIR: attenuated total reflection-Fourier transform infrared spectroscopy; ER: enhancement ratio; FBS: fetal bovine serum; FCM: flow cytometry; GC-MS: gas chromatography-mass spectrometry; HPLC: high-performance liquid chromatography; NMDA: N-methyl-D-aspartic acid receptor; OT: osthole; PBS: phosphate buffer saline; PO: paeonol; Q24: cumulative amount of drugs permeating through the skin at 24 h; SC: stratum corneum; SVO: white mustard seed volatile oil; TEM: transmission electron microscopy; Tlag: lag time; 5-FU: 5-Fluorouracil

ACCEPTED MANUSCRIPT Using gas chromatography-mass spectrometry, we showed that SVO was composed primarily of allylisothiocyanate and isothiocyanatocyclopropane. Compared with azone, SVO had better penetration-enhancing effects on three model drugs (5-Fluorouracil, Osthole, and Paeonol), with each having different oil-water partition

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coefficients. Histopathology showed that SVO did not induce skin irritation when the

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concentration was lower than 2% (v/v), and it induced less irritation than azone.

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According to attenuated total reflection-Fourier transform infrared spectroscopy and transmission electron microscopy, SVO induced skin lipid structural disorder and

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increased the distance between the stratum corneum, which is beneficial to the

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penetration of drugs. Cellular experiments showed that SVO inhibited Ca2+-ATPase activity, increased intracellular Ca2+ concentration, and changed the membrane

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potential in HaCaT cells, which promoted drug transfer into the skin. Our findings

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reveal that SVO is a safe and efficient natural product that has great potential as skin penetration enhancer.

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Key words: White mustard, Volatile oil, Natural Penetration Enhancer, Percutaneous

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penetration, Membrane Potential.

1. Introduction

Human skin consists of three parts: epidermis dermis, and subcutaneous tissue. It acts as a protective layer that guards the body from damage caused by the external environment [1]. However, the skin also acts as a barrier to percutaneous penetration of drugs. Transdermal administration is a drug delivery method with multiple

ACCEPTED MANUSCRIPT advantages, which include acting on the skin directly and allowing for absorption into the body through capillaries and lymphatic vessels. It is non-invasive and provides a large absorption surface area [2]. Furthermore, transdermal administration allows drugs to bypass the first pass effect. As skin metabolism of drugs is relatively low,

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most transdermal drugs exhibit zero-order kinetic metabolic profiles. Thus,

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transdermal administration allows for prolonged drug effects and less inter-subject

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variability.

However, transdermal delivery of drugs is often limited by poor permeability. The

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outermost layer of the skin, stratum corneum (SC), is the main rate-limiting layer to

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drug penetration into the skin [3, 4]. The SC is primarily composed of keratinocytes and intercellular lipids, which form a complex cross-interlocking structure [5]. Thus,

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only drugs with optimal physicochemical properties can be transported through the

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skin. Therefore, transdermal enhancers are necessary components in topical preparations. They temporarily weaken skin barrier properties and increase the

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percutaneous penetration rate, to promote transdermal permeation. Natural products,

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such as volatile oils, terpenes, fatty acids, and polysaccharides, are good candidates for use as transdermal enhancers, due to their relatively low toxicity, low irritation, and low allergenic potential, compared to synthetic chemicals[6]. Among these natural products, volatile oils are most likely to function as percutaneous penetration enhancers, as they interfere with the structure of the SC and interact with intercellular SC lipids to increase drug diffusion [7]. Recently, studies have shown that many volatile oils in traditional Chinese medicines

ACCEPTED MANUSCRIPT can promote drug absorption. Various plant species that emit volatile oils with transdermal permeation properties have been identified in the literature: clove (Ewgewia caryophyllata Thunb.) [8]; Sichuan pepper (Zanthoxylum bungeanum Maxim). [9]; black cardamom (Alpinia oxyphylla) [10]; anise (Foeniculum vulgare

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Mill. )[11]; and ginger (Zingiber officinale Rose.) [12]. Some volatile oils also have

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medicinal properties and exert therapeutic effects.

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The mature dry seeds of white mustard, Sinapis alba L., are used for the external treatment of joint numbness and deep abscess diseases in traditional Chinese medicine.

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White mustard seed volatile oil (SVO) comprises the active ingredients for its

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effectiveness [13, 14]. In China, acupoint application of herbal pastes for asthma is a renowned traditional therapy that uses transdermal drug delivery. S. alba plays a key

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role in this therapy, and the local skin reaction it causes correlates strongly with it

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therapeutic effectiveness [15, 16]. Because of these benefits, SVO is a promising agent to promote penetration of other drugs; however, its mechanism as a

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percutaneous penetration enhancer has not been reported. This study aimed to

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evaluate the potential of SVO as a natural transdermal penetration enhancer and characterize the possible mechanisms of penetration enhancement. 2. Materials and methods 2.1 Plant material and extraction of SVO The dried seeds of white mustard used in this work were purchased from by Kangmei Pharmaceutical Co., Ltd. (GuangDong, China) and identified by Prof. Xing-Xing Chan (School of Traditional Chinese Medicine, Southern Medical University, China).

ACCEPTED MANUSCRIPT The seeds were pulverized and placed in of disodium hydrogen phosphate-citrate buffer (pH 5), followed by the addition of ascorbic acid (0.5 mmol/L)( Sigma-Aldrich, USA). The mixture was hydrolyzed at 50 °C for 3 h, then extracted by steam distillation to obtain SVO, which is pale yellow in color.

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2.2 Gas chromatography-mass spectrometry analysis

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Gas chromatography-mass spectrometry analysis (GC-MS) analysis of SVO was

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performed using a Thermo Scientific™ Trace™ 1300 (Thermo Scientific, USA) for chromatography spectrometric characterization and quantitation of analytes eluted

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from the GC column. Analytes were separated using a DB-5ms column (Agilent, USA)

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in the temperature program. Mass spectra (Orbitrap Fusion™ Lumos™ Tribrid™ , Thermo Scientific, USA) were obtained by automatic scanning across the mass range

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of m/z 35-550. Spectra were searched in the NIST14 mass spectral database to

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identify SVO components. The percent composition of compounds (relative quantity) in SVO was calculated using normalized GC peak areas without correction factors.

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2.3 Permeation experiments

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Sprague Dawley (SD) rats were supplied by the Laboratory Animal Center, Southern Medical University. Animal experiments were performed in accordance with the principles of The Institutional Animal Care and Use Committee. Our study was approved by the Animals Ethics Committee of Southern Medical University. To determine the amount of drug deposited in the skin, we used Franz diffusion cells (Xin-Zhou Technology Co., Ltd, TianJin, China) to evaluate permeability. Skin obtained from male SD rats was clamped between the donor and the receptor

ACCEPTED MANUSCRIPT chambers, with the respective formulations 5-fluorouracil (5-FU, n-octanol/water partition coefficient: logKo/w = -0.95) [17]), paeonol (PO, logKo/w = 2.054) [18]), and osthol (OT, logKo/w = 3.85) [9] in 0.5%, 2%, and 5% SVO or azone, placed in the donor chamber. PEG400/saline was used as the base solvent. All three model

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drugs (5-FU, PO, and OT, Aladeen Co. Ltd (Shanghai, China)) were prepared in a

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saturated solution with a solvent of 30/70 (v/v) PEG400/saline. The model drugs were

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dissolved in the base solvent without penetration enhancers as the controls and with 0.5%, 2%, or 5% of either SVO or azone as the experimental groups. The Franz

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diffusion cells with mounted skin samples were placed in a water bath with a constant

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temperature of 32 °C equipped with a magnetic stirrer. The acceptor phase (30% PEG400/saline) was stirred at 32 °C throughout the experiment. The permeation

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membrane was dismantled after 1, 2, 4, 6, 9, 12, and 24 h. All samples were analyzed

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using the corresponding HPLC method described below. 2.4 HPLC analysis of model drugs

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HPLC analysis was carried out on an Agilent 1290 (Agilent, USA) HPLC equipped

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with an HC-C18 column (5 μm, 4.6 × 250 mm) (Waters, USA)and a diode array detector (DAD). For 5-FU analysis, the mobile phases consisted of 95% water and 5% methanol. The detection wavelength was set to 270 nm with the retention time of 5.0 min. The calibration curve was linear over the range 1–80 μg/ml (r2 = 0.9997). For OT and PO analyses, the mobile phases consisted of A (55% water and 45% methanol) and B (35% water and 65% methanol), respectively. The detection wavelengths were set to 322 and 276 nm for OT and PO, with retention times of 10.2 min and 7.3 min,

ACCEPTED MANUSCRIPT respectively. The calibration curve was linear over the range 0.1–200 μg/ml (r2 = 0.9996) of OT and 1–1000 μg/ml for PO (r2 = 0.9993). The injection volume was set to 20 μL, and flow rate at 1 mL/min. 2.5 Attenuated total reflection-Fourier transform infrared spectroscopy studies

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Rat skin was placed in a 0.4% trypsin (Sigma-Aldrich, USA) solution and kept at

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room temperature for 10 h. Then, the skin cuticle was carefully separated with a

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cotton swab, washed with distilled water, and dried in a vacuum oven. Thereafter, 1 × 1 cm2 pieces of dry SC were treated in 5 mL solvent with either (0.5%, 2%, 5%) (v/v)

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SVO/saline or (0.5%, 2%, 5%) (v/v) azone/saline at room temperature for 12 h. The

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SC sheets were cleaned carefully with distilled water and dried in a vacuum oven at 37 °C. The dry SC sheets were measured by attenuated total reflection-Fourier

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transform infrared (ATR-FTIR) spectroscopy under a resolution of 2 cm-1, 100 scans,

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and scanning range of 650–4000 cm-1. 2.6 Transmission electron microscopy

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The blank group (not treated), control solution group (saline), azone groups (0.5%,

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2%, and 5% azone), and SVO groups (0.5%, 2%, and 5% SVO) were evaluated by transmission electron microscopy (TEM). Rat skin was cut into small pieces and treated with the different solutions mentioned above for 12 h at 37 °C. Thereafter, pieces of rat skin were cleaned with 0.1 mol/L phosphate buffered saline (PBS, pH 7.3) and fixed in 2.5% glutaraldehyde/PBS (w/v) at 4 °C overnight. The skin was rinsed with 0.1 mol/L phosphate buffer (pH 7.3) for 15 min each time for 3 times. The rinsed skin was placed in 1% citric acid and fixed for 2 h, then washed with 0.1 mol/L

ACCEPTED MANUSCRIPT phosphate buffer. For dehydration, we used the protocol per: 50% acetone 20 min, 70% acetone 20 min, 80% acetone 20 min, 90% acetone 20 min, 100% acetone 10 min, and 100% acetone 10 min. For soaking and embedding, we placed the skin in the solvent with 50/50 (v/v) acetone/embedding buffer (GENMED, Shanghai, China),

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baked it at 37 °C for 2 h; and placed it in 20/80 (v/v) acetone/embedding buffer at

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37 °C overnight; then it was placed in pure embedding buffer at 45 °C for 2 h. The

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skin-embedded block was placed in an oven at 45 °C for 12 h, baked at 65 °C for 72 h, then dried. Sectioning was performed using an ultramicrotome (Leica EM UC7,

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Germany). For dyeing, ultrathin sections were loaded onto a copper mesh and stained

(Hitachi H-7500, Hitachi, Japan).

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2.7 Skin irritation studies

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with uranyl acetate-lead citrate for 20 min. Sections were visualized under a TEM

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SD rats were randomly divided into eight groups according to sex and weight: blank; control; 0.5%, 2%, and 5% SVO; and 0.5%, 2%, and 5% azone. All rats were

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depilated on both sides of the back spine for 24 h before application [19-21]. The

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experiment was performed by the self-control method. The left skin of the rats was not treated (blank group) or treated with saline (control group). The right skin was exposed to different concentrations of SVO or azone in saline. The test article was applied daily for 4 h, once per day, for 7 consecutive days. At the end of the last application, the skin was excised, immersed in a 10% formaldehyde solution, and then embedded in paraffin for fixation. The wax block was cut to a thickness of 3–5 μm and stained with hematoxylin-eosin (Sigma-Aldrich, USA)for histopathological

ACCEPTED MANUSCRIPT examination (scale ×200). 2.8 Cell line and culture Human skin epidermal keratinocytes (HaCaT) were supplied by Nanfang Hospital Central Laboratory at Southern Medical University (Figure 4A). HaCaT were

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incubated in complete medium, contained Dulbecco's Modified Eagle’s medium

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(DMEM, Gibco, USA), 10% fetal bovine serum (FBS, Gibco, USA) and 100 U/mL

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penicillin/streptomycin (Gibco, USA) in a 5% CO2 incubator at 37 °C. 2.9 Cytotoxicity assay

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A CCK-8 kit was used to determine the cellar toxicities of SVO and azone. HaCaT

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cells were collected and inoculated at a cell density of 1 × 104 cells per well. After 24 h of incubation, groups were exposed to SVO or azone at different concentrations, in

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0.1% dimethyl sulfoxide (DMSO) for 24 h. Cell viability was determined according to

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the CCK-8 kit method. According to the cell survival rates of different groups, we calculated the drug concentrations required to induce 50% inhibition.

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2.10 Concentration of Ca2+ in HaCaT cells

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HaCaT cells were inoculated in 25 mL culture flasks for 12 h and placed into six groups: blank group (complete medium), control group (0.1%DMSO), SVO groups (0.16, 0.32, and 0.64 mg/mL SVO), and azone group(0.16 mg/mL azone). After incubating for 24 h, HaCaT cells were digested into monoplasts and adjusted to 7.0 × 105 cells/mL by adding PBS. The monoplasts were separated from the media, and the fluorescent probe Fluo3-AM (500 μL, 5 µM final concentration) was added. The cells were then incubated for 0.5 h. Fluorescence intensity was measured using flow

ACCEPTED MANUSCRIPT cytometry (FCM), with excitation and emission wavelengths of 488 and 520 nm, respectively. HaCaT cells without fluorescent labels were used for zero adjustment. 2.11 Enzymatic activity of Ca2+-ATP in HaCaT cells HaCaT cells were inoculated and placed into six groups as described in Section 2.10.

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After incubating for 24 h, HaCaT cells were digested into monoplasts and adjusted to

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4.5 × 106 cells/mL by adding PBS. The cells were fully ground using an ultrasonic

ultramicro-Ca2+-ATP

detection

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cell disruptor, and Ca2+-ATP enzymatic activity was determined using an kit

Institute

of

Biological

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Engineering ,NanJing, China).

(Nanjing

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2.12 Membrane potential of HaCaT cells

HaCaT cells were inoculated and placed into six groups per Section 2.10. After

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incubating for 24 h, HaCaT cells were digested into monoplasts and the media were

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removed from the monoplasts. Stock solution of a fluorescent probe, DiBAC4 (3) (500 μL, final concentration 5 µg/mL, Invitrogen,USA) was added. The cells were

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incubated for 0.5 h. The monoplasts were separated from the solvent by centrifugation

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and dispersed with PBS. Fluorescence intensity was measured using FCM. Excitation and emission wavelengths were 488 and 530 nm, respectively, and unlabeled HaCaT cells were used for zero adjustment. 2.13 Data and Statistical analysis All experimental data were statistically analyzed using SPSS 20.0 software, and statistical results of measurement data were expressed as mean ± standard deviation (X

 SD

). Statistical differences were tested by one-way analysis of variance

ACCEPTED MANUSCRIPT (ANOVA) and the independent samples t-test. P < 0.05 was considered significant. Transdermal permeation kinetic curves of each drug were obtained according to cumulative permeation per unit area of each model drug plotted against time. Q (μg/cm2) is the cumulative amount of drug permeated across the skin. Q24 is the

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cumulative amount of drug permeated across the skin over 24 hours. The linear

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portion was subjected to linear regression to find the slope of the line J

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[μg/(cm2 · h)], and flux was calculated from the slope of the linear portions of the

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curves. The lag time (Tlag) was determined by extrapolating the linear portion of the curve to the X-axis. The enhancement ratio (ER) was used to compare the ability of

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the permeation enhancers: ER = flux for skin treated with the enhancer/flux for the control.

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3. Results

3.1 Chemical composition of SVO

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The chemical substances and relative contents of SVO detected by GC-MS (Figure 1) are listed in Table 1. Fourteen compounds were identified in SVO. The total peak area

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of the chromatogram minus the solvent peak was used as 100% total area of the chromatogram, and the relative content of each chemical component in the sample was determined by normalization. The major compounds were 3-butenenitrile (16.62%), allylisothiocyanate (57.02%), and isothiocyanatocyclopropane (17.46%). The main components generally determine the biological properties of volatile oils. Allylisothiocyanate can active Ca2+ influx into intracellular, which may promote percutaneous penetration[22, 23].

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Tab. 1 components of SVO by GC-MS

Retention

Molecular

No.

Molecular

Relative

weight

contents(%)

Compounds formula

1

4.250

C4H5N

3-butenenitrile

67.09

16.62

2

6.033

C5H7N

3-methylbut-2-enenitrile

81.12

0.23

3

7.585

C4H5NS

allylisothiocyanate

4

8.069

C4H5NS

isothiocyanatocyclopropane

5

9.257

C5H7NS

4-isothiocyanatobut-1-ene

6

10.285

C6H11NS

7

10.328

8

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time

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99.15

57.02 17.46

113.181

1.45

1-isothiocyanato-2-methylbutane

129.23

0.13

C6H11NS

1-isothiocyanato-3-methylbutane

129.22

0.14

12.064

C5H12S

isobutyl(methyl)sulfane

104.214

0.17

9

14.339

C9H9N

Benzenepropanenitrile

131.17

1.04

10

14.790

C5H9NS2

(3-isothiocyanatopropyl)(methyl)sulfane

147.26

2.16

11

15.377

C3H6S4

5-Methyl-1,2,3,4-tetrathiane

170.34

0.24

12

16.740

C9H9NS

(2-isothiocyanatoethyl)benzene

163.24

2.91

13

17.806

C15H18O2

Epicurzerenone

230.30

0.28

14

18.045

1,2,4,5-tetrafluoro-3-methoxybenzene

180.10

0.16

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SC

99.15

C7H4F4O

3.2 Effects of SVO on percutaneous absorption of model drugs To investigate the permeation enhancement mechanism, rat skin is a well-accepted model [17, 24, 25]. All model drugs and enhancers had good solubility in the base solvent. The percutaneous profiles and percutaneous permeation parameters of different

ACCEPTED MANUSCRIPT concentrations of SVO on the model drugs are shown in Tables 2–4 and Figure 2, respectively. Our results showed that SVO had obvious permeation-enhancing effects on the three model drugs compared to those of the controls. Increasing the permeation enhancer concentration resulted in higher flux and Q24, demonstrating a

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concentration-dependent effect. Except for OT, increasing concentrations of SVO

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significantly increased Tlag compared with those of the blank group (P < 0.05).

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Compared with the control, the Tlag value for OT did not change markedly with changing SVO concentrations. SVO may effectively increase the permeation rate and

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the cumulative penetration of hydrophilic and moderately polar drugs, but it also

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resulted in a longer Tlag. For more lipophilic drugs, SVO had similar permeation-enhancing effects, but little effect on Tlag. SVO is comprised of lipophilic

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agents, which may be of more benefit to lipophilic drugs for penetration. Further

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experimental exploration is needed to elucidate the mechanism. The penetration effects of SVO on the model drugs differed per polarity: medium polarity >

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hydrophilic > lipophilic.

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Azone also enhanced penetration of the three model drugs. However, from ER results at equivalent concentrations, SVO showed better penetration enhancement than azone. Moreover, the ability of azone to promote permeability was different from SVO, regarding polar selectivity: hydrophilic > medium polarity > lipophilic. This suggests that different types of penetration enhancers may be selective for drugs with different oil-water partition coefficients. Choosing a suitable penetration enhancer can better achieve the goal of promoting penetration [9]. Despite this, SVO has greater

ACCEPTED MANUSCRIPT potential than azone as penetration enhancer. However, to accurately evaluate the enhancement properties of SVO, further experiments using human skin are required. Tab. 2 Percutaneous permeation parameters of 5-FU through excised rat skin

Penetration

Concentration

Flux

Tlag

Q24 ER

(μg·cm ·h )

(h)

control

0.39±0.02

0.59±0.06

0.5%

1.06±0.14*

2.89±0.21*

2%

1.71±0.28*

5%

2.89±0.43*

control

0.66±0.01

0.5% 2%

9.28 ±3.59

/

25.59 ±5.88*

2.72

3.51±0.26*

41.45 ±4.24*

4.39

3.50±0.30*

68.88 ±12.35*

7.40

0.43±0.05

17.44 ±6.51

/

1.44±0.19*

3.74±0.18*

34.99 ±8.63*

2.17

2.09±0.26*

4.06±0.31*

44.62 ±5.72*

3.15

6.79±0.55*

59.96 ±6.37*

5.23

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SVO

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Azone

3.47±0.19*

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5%

(μg·cm )

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(v/v)

-2

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-1

SC

enhancer

-2

Values are expressed as the means with S.D. (n = 6). *Indicates statistically significant difference in comparison to

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the control at p< 0.05.

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Tab. 3 Percutaneous permeation parameters of OT through excised rat skin

Penetration enhancer

Concentration

Flux

Tlag

Q24 ER

-2

-1

-2

(v/v)

(μg·cm ·h )

(h)

(μg·cm )

control

1.66±0.05

3.48±0.47

34.53 ±3.38

/

0.5%

2.20±0.07*

3.64±0.33

44.95 ±2.91*

1.33

2%

3.79±0.31*

2.84±0.19

87.22 ±18.26*

2.28

5%

7.61±0.71*

3.03±0.17

175.55 ±25.30*

4.59

SVO

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control

1.64±0.05

3.26±0.36

33.64 ±8.72

/

0.5%

2.08±0.12*

2.68±0.25*

43.55 ±6.68

1.27

2%

2.74±0.10*

3.00±0.18*

56.51 ±15.35*

1.68

5%

5.36±0.29*

3.38±0.22*

113.90 ±17.29*

3.28

Azone

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Values are expressed as the means with S.D. (n = 6). *Indicates statistically significant difference in comparison to

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the control at p< 0.05.

(v/v)

(μg·cm ·h )

(h)

(μg·cm )

control

5.07±0.06

0.84±0.11

117.84 ±18.87

/

0.5%

10.66±0.96*

2.16±0.17*

254.23 ±33.65*

2.10

2%

41.10±1.18*

8.20±1.05*

650.52 ±48.89*

8.10

59.65±1.08*

7.53±0.94*

983.89 ±86.21*

11.76

control

2.70±0.44*

1.43±0.20*

99.38±10.54

/

0.5%

4.63±0.29*

1.07±0.21*

176.17±25.53*

1.71

2%

6.14±0.27*

0.7±0.09*

244.56±21.67*

2.27

5%

11.76±0.28

0.47±0.10*

437.59±33.31

4.35

Concentration

-2

-1

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Azone

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SVO

5%

Tlag

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enhancer

Flux

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Penetration

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Tab. 4 Percutaneous permeation parameters of PO through excised rat skin

Q24 ER -2

Values are expressed as the means with S.D. (n = 6). *Indicates statistically significant difference in comparison to

the control at p< 0.05.

3.3 ATR-FTIR spectroscopy studies The main components of the SC are lipids and keratin. FTIR (Tensor II, BRUKE, Germany) was be used to obtain absorption peak information of SC lipid and keratin

ACCEPTED MANUSCRIPT secondary structures, allowing for evaluation of changes in its molecular structure. Shifts in these peaks indicate structural changes. Decreases in the spectral peak area indicate decreased content of the associated components in the SC [26]. These results are summarized in Figure 3A and Table 5.

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Tab.5 Peak positions of C-H stretching of lipids and amides treated with agents

Groups

Symmetric C-H

SC

Asymmetric C-H

Peak position of SC(cm-1)

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Peak position of lipid(cm-1)

Keratin amide I

stretching

Blank

2916.87

Control

2916.74

0.5%Azone

2918.40

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stretching

Keratin amideⅡ

1644.66

1539.12

2849.21

1644.09

1538.94

2850.28

1643.57

1538.72

2919.68

2851.33

1644.14

1538.22

2921.33

2851.67

1644.35

1538.68

2917.19

2849.43

1644.47

1539.57

2% SVO

2918.96

2850.53

1643.82

1538.64

5% SVO

2921.36

2851.84

1644.55

1538.35

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D

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2%Azone

AC

CE

5%Azone

0.5%SVO

2849.21

# Blank: no treated, control: saline, SVO/azone: penetration enhancer dissolved in saline

Our results show that when applied with azone, the symmetric vibration absorption peak and asymmetric vibration peak of SC lipid C-H shifted to higher wavelengths, with significant effects observed in response to 0.5% azone. When the concentration

ACCEPTED MANUSCRIPT reached 5%, the asymmetric vibration absorption peak of lipid C-H and the symmetric vibration absorption peak of lipid C-H shifted by approximately 4 cm-1 and 2 cm-1, respectively. When applied with SVO, the symmetric vibration absorption peak and the asymmetric vibration peak of SC lipid C-H shifted by approximately 4 cm-1 and 3

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cm-1, respectively, similar to the action of azone. However, SVO and azone had no

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significant effect on Keratin amide I and Keratin amide II absorption peaks of the SC,

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indicating that SVO and azone had little effect on keratin in the SC. These results suggest that SVO and azone can change the molecular structure of the SC and extract

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lipids, thereby affecting the barrier function of the SC.

3.4 Morphology of the cuticle in response to transdermal enhancers

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Changes in the SC of rats treated with different concentrations of azone and SVO are

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shown in Figure 3B. The SC of normal skin is distributed in a strip on the outermost layer of the skin, and the inter-layer distance is also tight, exerting an effective barrier

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(Figure 3B(a)). Compared with the blank group, the number of SC layers and the layer

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gaps in the control group did not obviously change (Fig. 3B(b)), suggesting that saline did not affect SC morphology. Figure 3B(f, g, h) and 3B(c, d, e) show morphological changes of rat skin SC in response to azone (0.5%, 2%, and 5%) and SVO (0.5%, 2%, and 5%), respectively. There were no significant changes in the number of SC layers in response to 0.5% azone or SVO, but the interlayer distance increased. With increasing concentration of penetration enhancers, the interlayer distance further increased, and the number of SC layers decreased significantly. When the

ACCEPTED MANUSCRIPT concentration reached 5%, the SC structure was severely damaged and substantially peeled off. SVO induced concentration-dependent changes in SC morphology, consistent with its effects on the permeation of model drugs in vitro. These results further highlight the important role of the SC as a protective barrier. Although higher

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concentrations of penetration enhancers had a relatively high penetration-enhancing

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effect, a significant amount of damage was done to the SC structure, which may cause

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skin irritation or toxicity.

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3.5 Skin irritation

The evaluation of skin irritation in response to drug penetration enhancers is essential.

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An excellent penetration enhancer should be safe and non-irritating to the skin. Our results are summarized in Figure 3C.

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There were no significant differences in skin morphology between the control (Figure

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3C(e)) and blank (Figure 3C(a)) treatments, indicating that saline did not irritate the skin. Following application of 0.5% and 2% SVO (Figure 3C(b, c)) or azone (Figure

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3C(f, g)) to the skin, no inflammation, edema, or tissue necrosis was observed. After 7

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days of 5% azone (Figure 3C(h)) or SVO (Figure 3C(d)) application, inflammatory cell infiltration was observed; and the dermis was congested with edema, the epidermis partially fell off, and slight inflammatory cell infiltration was observed. At higher concentrations (5% v/v), the level of irritation does not justify the increased permeation enhancement, resulting in these concentrations being unsuitable for use. Lower concentrations (< 2% v/v) of penetration enhancers were less irritating to the skin and resulted in no histopathological changes, indicating that SVO at a suitable

ACCEPTED MANUSCRIPT concentration for transdermal permeation did not result in skin irritation. 3.6 Toxicity of SVO in HaCaT cells Toxicity of penetration enhancers was examined using CCK-8 assay in HaCaT cells.

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After 24 h of treatment with SVO or azone, cell viability decreased in a dose-dependent manner (Figure 4 B and C). The IC50 values of azone and SVO were

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0.2621 ± 0.017 mg/mL and 1.5906 ± 0.032 mg/mL, respectively. Cell viability of

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HaCaT cells was higher when the concentrations of azone and SVO were lower than

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0.16 mg/mL and 0.64 mg/mL, respectively. Therefore, SVO at concentrations of 0.16, 0.32, and 0.64 mg/mL were selected as low, medium, and high doses to investigate the

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effects of Ca2+ concentration, Ca2+-ATPase activity, and membrane potential on

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HaCaT cells.

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3.7 Effect of SVO on Ca2+ concentration in HaCaT cells Ca2+ plays an important regulatory role in physiological and biochemical reactions

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[27]. If intracellular and extracellular Ca2+ balance is disrupted, membrane fluidity of keratinocytes and morphology of tight junctions, and membrane potential will be

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disturbed, affecting drug permeability and the overall barrier function of the skin. Flow cytometry was used to determine the effects of different doses of SVO and 0.16 mg/mL azone on Ca2+ concentrations in HaCaT cells. Both SVO and azone increased Ca2+ concentrations in HaCaT cells. Fluorescence intensity after treatment with SVO is shown in Figure 4D and E. Cells treated with SVO showed a significant increase in intracellular Ca2+ concentration, and fluorescence intensity increased in a

ACCEPTED MANUSCRIPT dose-dependent manner. There were no significant differences in fluorescence intensity in the low-dose SVO group compared to the control group. These results showed that SVO increased Ca2+ concentration in HaCaT cells and resulted in penetration activity similar to that observed with azone.

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3.8 Effect of SVO on activity of Ca2+-ATPase in HaCaT cells

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As intracellular Ca2+concentration is mainly regulated by Ca2+-ATPase, when

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intracellular Ca2+-ATPase activity is reduced, Ca2+ cannot be pumped out of the cell.

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This indirectly increases intracellular Ca2+ concentration, which may alter membrane fluidity and intercellular junctions of keratinocytes, causing the intercellular space to

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increase.

Intracellular Ca2+-ATPase activity in each SVO group significantly decreased

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compared to that of the control group (P < 0.05). As the concentration of SVO

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increased, intracellular Ca2+-ATPase activity decreased (Figure 4H). This indicates

activity.

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that SVO can exert its penetration action by reducing intracellular Ca2+-ATPase

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3.9 Effect of SVO on Membrane Potential of HaCaT cells The presence of an ion pump in the cell membrane maintains an ion concentration gradient inside and outside the cell to form a membrane potential. Changes in membrane potential are accompanied by changes in cell membrane function and structure, such as cell membrane ion permeability. There is a fixed negative charge at the junction of skin keratinocytes [28]. Previous studies have shown that when some transdermal penetration enhancers interact with these negative charges, they can

ACCEPTED MANUSCRIPT promote transport of drugs through the skin [29]. The results of SVO and azone on membrane potential of HaCaT cells are shown in Figure 4F and G. As DiBAC4 (3) is negatively charged, greater fluorescence intensity is associated with lower membrane potential. Low concentration SVO had no obvious

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effect on the membrane potential of HaCaT cells (P > 0.05). However, increasing

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SVO concentrations significantly enhanced fluorescence intensity. These results

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showed that SVO may increase permeation and absorption of the drug by decreasing

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the membrane potential of HaCaT cells. 4. Discussion

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As previously discussed, the SC is composed of keratinocytes and intercellular lipids, forming a cross-interlocking structure. This structure is the main barrier to

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percutaneous penetration of most drugs. Drug components with moderate polarity easily pass through the active epidermis, and drug components with high lipophilicity

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or high hydrophilicity do not penetrate readily. Therefore, skin penetration enhancers are necessary to increase transdermal permeability of topical preparations. Exploring

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the interaction between skin penetration enhancers and components in the SC of the skin plays a crucial role in explaining the mechanisms of penetration enhancers. There are many ways to promote transdermal drug delivery. The chemical penetration-enhancing approach is to increase penetration of the drug into the skin by altering the physicochemical properties of the drug molecule or the SC, including preparation of penetration enhancers and prodrugs. The physical promotion method

ACCEPTED MANUSCRIPT refers to promotion of drug penetration by physical means, including ultrasonic introduction, iontophoresis, and microneedle [30]. The pharmaceutics method refers to use of drug carrier technology to promote percutaneous absorption of a drug with poor skin permeability by means of a carrier with good permeability that can carry the

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drug, such as liposomes or microemulsions [31]. The use of skin penetration

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enhancers is the most widely used mechanism to increase skin permeation.

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SVO is a natural product that has good penetration-promoting ability and results in

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little irritation in the effective concentration range. Thus, it has great potential as a transdermal absorption enhancer. In the present study, HaCaT cells, which account for

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approximately 95% of the skin [32], were used as a model to evaluate the mechanisms by which SVO promotes skin penetration, with azone, a type of transdermal

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penetration enhancer widely used in the pharmaceutical and cosmetic industries, as a

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positive control for comparison [33, 34].

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Our in vitro transdermal experiments showed that SVO exerted penetration effects on the model drugs with different oil-water partition coefficients, with the effect on

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medium-polarity drugs being the most significant. At an equivalent concentration to azone, SVO was more effective in promoting permeation of the model drugs than azone. In vivo animal experiments also showed that SVO was less irritating to the skin than azone. Compared with azone, SVO has potential as an excellent skin penetration enhancer. Generally, transcellular pathway and intercellular lipid pathway were considered as

ACCEPTED MANUSCRIPT the main pathways for drug penetration through the skin. The structure, state, and conformation of the lipid components and keratin of the skin change, and the structure and conformation of the SC of the epidermis also changes, resulting in skin permeability and barrier function changes [35]. ATR-FTIR was used to explain the

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effects of penetration enhancers on lipid molecules and keratin molecules in the SC of

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the skin, allowing for accurate evaluation of changes in the SC related to

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penetration-enhancement in the skin. The blue shift of asymmetric C-H and symmetric C-H usually suggested a more disordered organization of SC intercellular

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lipid. Skin treated with SVO changed the lipid conformation in the SC, and lipid

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disorder increased, which destroyed the ordered and dense membrane structure of the SC and increased skin permeability. Increased permeability promoted penetration of

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the drug into the skin, resulting in the penetration action of SVO. The morphology of

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the lipid structure and SC structure of the skin was observed by TEM to determine the effect of SVO on the skin barrier. Observation of the microstructure of the SC in

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response to SVO by light microscopy and TEM showed disruption of the ordered and

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dense membrane structure of the SC, and cell gaps became larger. The interlaminar space of the SC was enlarged, the structure became loose, and skin permeability increased, which was beneficial to drug penetration. There are two sources of intracellular calcium ions. The first source is extracellular Ca2+ that enters the cell through calcium channels on the cell membrane, and the other is from intracellular calcium pools. Phospholipase C on the cell membrane is activated to hydrolyze inositol lipids, resulting in intracellular increases in creatine triphosphate,

ACCEPTED MANUSCRIPT prompting the calcium pool to release Ca2+. Ionotropic glutamate receptors of the N -methyl- d -aspartate (NMDA) receptor type and transient receptor potential A1(TRPA1) could enable a transmembranous calcium influx from the extracellular space. Increased Ca2+ concentration in human keratinocytes can cause contraction of

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actin filaments and contraction of cells [22], increasing intercellular space and skin

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permeability, allowing drugs to penetrate the skin more easily. This study found that

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SVO can increase the Ca2+ concentration in HaCaT cells, which is beneficial to increase the skin permeability of model drugs. The allylisothiocyanate mainly

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contained in SVO can cause the activation of TRPA1, which causes Ca2+ influx into

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cellular[23]. Although the activation of TRPA1 by allylisothiocyanate has been thought to be associated with triggering pain. However, in the case of low doses of

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SVO, combined with the results of skin irritation, we believe that SVO may cause an

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increase in intracellular calcium concentration in HaCaT by TRPA1 activation without triggering skin irritation. Besides, the activation of calcium permeable ionotropic

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channels on the epidermal keratinocytes induced epidermal barrier abnormality[36].

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Topical application of an NMDA receptor agonist delayed barrier recovery[37]. The activity of NMDA involved in the regulation of calcium influx into intracellular has been related to the expression of Ca2+-ATPase[38]. SVO can inhibit the outflow of calcium ions from intracellular by inhibiting Ca2+-ATPase. Calcium increased in epidermal keratinocytes that delayed the recovery of barrier function after barrier disruption[39] and promote skin penetration. In addition, increased transepidermal water loss (TEWL) in damaged skin may be related to calcium ion influx in

ACCEPTED MANUSCRIPT keratinocytes[40]. Experiment have shown that the application of azone as a penetration enhancer on the skin increases the TEWL[41]. Increasing TEWL may also be one of the ways that azone promote skin penetration, but it is also an essential factor in triggering skin irritation. These evidences suggest that calcium ions influx

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into keratinocytes and weaken skin barrier function, which is beneficial for

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percutaneous penetration of drugs. In addition, if the balance of intracellular and

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extracellular Ca2+ concentrations is disrupted, the membrane potential of the cell changes, and the permeability of the drug changes. The morphological structure of

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epidermal cells and the change in membrane potential affect transdermal drug

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transport. The decrease in cell membrane potential of HaCaT interfered with by SVO increased cell membrane fluidity to promote penetration.

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To sum up, SVO facilitate the drug penetration by increasing the mobility and the

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disordered organization of SC intercellular lipid, which was proved by the results of ATR-FTIR and TEM. On the other hand, SVO may be due to decreased intracellular

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Ca2+-ATPase activity and increased Ca2+ concentration in HaCaT cells via NMDA or

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TRPA1, resulting in changes in cell membrane potential, promoting absorption of the drug to deeper parts of the skin. 5. Conclusions Our study provided new insight into the interactions between SVO and skin barrier function, and we elucidated the possible penetration-enhancing mechanisms. Thus, SVO may be effective as a new type of low-toxicity and high-efficiency skin

ACCEPTED MANUSCRIPT penetration enhancer. Appendices Fig1. Total ionization mode of SVO.

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Fig2. Permeation profiles of three model drugs through excised rat skin with SVO and

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Azone. a,d - Fluorouracil (5-FU), b,e- Osthole(OT), c,f- Paeonol(PO); a,b,c with

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volatile oi; d,e,f with Azone

Fig3. Effects of penetration enhancers on skin microstructure. (A) FTIR spectra of SC

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after treated by different agents. (B) TEM images of rat stratum corneum after

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treatment with penetration enhancers. (a-Blank; b-Control; c-0.5% SVO ; d-2% SVO ; e-5% SVO ; f-0.5%Azone; g-2%Azone; H-5%Azone). (C) Effects of penetration

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enhancers on the morphology of rat skin. ((a)-Blank. (2)-Control(Saline).

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(b),(c),d)-0.5%,2%,5% SVO. (f),(g),(h)- 0.5%,2%,5% Azone. Fig4. Effects of penetration enhancers on the HaCaT. (A) Human skin epidermal

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keratinocytes (HaCaT) cell lines. (B) Effects on cell viability of HaCaT of azone. (C) HaCaT of different SVO. (D) Effects on Ca2+

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Effects on cell viability of

concentration of HaCaT of different transdermal enhancers. (E) Effects on fluorescence intensity of HaCaT of different transdermal enhancers. *P<0.05， **P<0.01. statistically significant difference between enhancers and blank. (F) Effects on cell membrane potential of HaCaT of different transdermal enhancers. (G) Effects on fluorescence intensity of HaCaT cell membrane potential of different transdermal enhancers. *P<0.05，**P<0.01. statistically significant difference between enhancers

ACCEPTED MANUSCRIPT and Blank. (H) Effects on Ca2+--ATPase activity of HaCaT of different transdermal enhancers. *P<0.05，**P<0.01. statistically significant difference between enhancers and blank.

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Acknowledgments This study was supported by the National Natural Science Foundation of China

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(81573611), National Natural Science Foundation of China (81573611), Natural

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Science Foundation of Guangdong Province (2017A030310021),the Science and

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Technology Program of Guangdong Province (2017A050506027) and the Science and

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Technology Program of Guangzhou (201807010053).

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Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence

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the work reported in this paper-“Mechanisms of white mustard seed

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(Sinapis alba L.) volatile oils as transdermal penetration enhancers”.

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Graphical abstract.

Figure 1

Figure 2

Figure 3

Figure 4