Decursinol angelate ameliorates 12-O-tetradecanoyl phorbol-13-acetate (TPA) -induced NF-κB activation on mice ears by inhibiting exaggerated inflammatory cell infiltration, oxidative stress and pro-inflammatory cytokine production

Article abstract of DOI:10.1016/j.fct.2019.110699

Decursinol angelate (DA) is a pyranocoumarin purified from the roots of Angelica gigas. Here, we synthesized DA and determined its anti-inflammatory potential on TPA-induced mice ear inflammation. First, we evaluated the non-toxic behaviour of DA on HaCaT cells. Additionally, we observed the free radical scavenging potential of DA at 60 μM to be 50%. This finding was further supported by nitric oxide assay, malondialdehyde assay, H2DCFDA staining and western blotting analysis of antioxidant enzymes. DA also suppressed the activation and polarization of macrophage phagocytic activity on RAW 264.7 cells. We further evaluated the expression of ICAM-1, MCP-1, MIP-2 and MIP-1β on in-vivo model system. Consequently, DA significantly reduced the production of NF-κB and COX-2 induced proinflammatory cytokine levels on TPA induced ear edema. Inhibition of MAPK and transcriptional factor NF-κB was also validated by western blotting analysis of p-ERK, p-p38, IKKα, IKKγ, IκBα, NF-κB-p65. Immunohistochemistry and immunofluorescence staining of NFκB-p65, TNF-α and IL-1β were also performed to support the findings. Conclusively, these results suggest that topical administration of DA significantly inhibited the expression of pro-inflammatory cytokines by blocking the canonical NF-κB and MAPK pathway. Therefore, we suggest DA as a potent therapeutic compound against skin inflammation related diseases.

Full text of DOI:10.1016/j.fct.2019.110699

Accepted Manuscript
Decursinol angelate ameliorates 12-O-tetradecanoyl phorbol-13-acetate (TPA) -
induced NF-κB activation on mice ears by inhibiting exaggerated inflammatory cell
infiltration, oxidative stress and pro-inflammatory cytokine production
Sukkum Ngullie Chang, Imran Khan, Debasish Kumar Dey, Kiu-Hyung Cho, Buyng
Su Hwang, Ki Beom Bae, Sun Chul Kang, Jae Gyu Park
PII:
S0278-6915(19)30489-2
DOI:
https://doi.org/10.1016/j.fct.2019.110699
Article Number: 110699
Reference:
FCT 110699
To appear in: Food and Chemical Toxicology
Received Date: 21 June 2019
Revised Date: 17 July 2019
Accepted Date: 23 July 2019
Please cite this article as: Chang, S.N., Khan, I., Dey, D.K., Cho, K.-H., Hwang, B.S., Bae, K.B., Kang,
S.C., Park, J.G., Decursinol angelate ameliorates 12-O-tetradecanoyl phorbol-13-acetate (TPA) -
induced NF-κB activation on mice ears by inhibiting exaggerated inflammatory cell infiltration, oxidative
stress and pro-inflammatory cytokine production, Food and Chemical Toxicology (2019), doi: https://
doi.org/10.1016/j.fct.2019.110699.
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ACCEPTED MANUSCRIPT
Decursinol angelate ameliorates 12-O-tetradecanoyl phorbol-13-acetate (TPA) -induced
NF-κB activation on mice ears by inhibiting exaggerated inflammatory cell infiltration,
oxidative stress and pro-inflammatory cytokine production
Sukkum Ngullie Changa,1, Imran Khanb,1, Debasish Kumar Deya, Kiu-Hyung Choc, Buyng Su Hwangd,
Ki Beom Baee, Sun Chul Kanga*, Jae Gyu Parke,*
aDepartment of Biotechnology, Daegu University, Gyeongsan, 38453, Republic of Korea
bDepartment of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha
University, 100 Inha-ro, Nam-gu, Incheon, 22212, Republic of Korea
cResearch group, Gyeongbuk Institute for Bio Industry(GIB), Andong, 36728, Republic of
Korea
dNakdonggang National Institute of Biological Resources, Sangju, 37242, Republic of Korea
eAdvanced Bio Convergence Center, Pohang Technopark Foundation, Pohang, Gyeongbuk
37668, Republic of Korea
*Corresponding authors at: Department of Biotechnology, Daegu University, Gyeongsan,
38453, Republic of Korea
Advanced Bio Convergence Center, Pohang Technopark Foundation, Pohang, 37668,
Republic of Korea
1Contributed equally to this work.

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Introduction
Inflammation is a major driver of many diseases. It is often caused through the response of
the immune system towards any stimuli it considers a threat, such as infectious micro-
organisms, toxic compounds, irradiation or even the host’s own damaged cell (Nathan C.,
Ding A., 2010). Different epidemiological studies have evidenced that inflammatory
mediators are the major player having crucial role during the development of various diseases
(P Libby et al , 2002; Korhonen et al., 2005; G S. Firestein., 2003) such as vascular diseases
(G W. Sullivan et al., 2000), neurological diseases (D Degan et al.,2018), chronic
inflammatory diseases (Rainer H. Straub et al.,2016) and even in many different types of
cancer (S I. Grivennikov et al., 2010). Whenever the immune system is challenged through
some external stimuli, the first line of defence i.e innate immunity comes in role, such as
macrophage activation. They are involved in the production many different types of pro-
inflammatory cytokines. Among those produced by M1 macrophages are IL-1β, IL-4, IL-6,
IL-12 and TNF-α While, M2 macrophage produces IL-10 and also very low levels of IL-12
(Mosser D.M., Edwards J.P., 2008). Nitric oxide (NO) is also one of the main mediators of
inflammation which stimulates the macrophage activation (J MacMicking et al., 1997).
However, nuclear factor kappa B (NF-κB) is the primary signalling pathway involved in the
process of initiating and amplifying inflammation (Li Q., Verma I.M. , 2002; Q Zhang et al.,
2017). NF-kB is a paradigm of rapid response factor that comes out of the latency from the
cytoplasm in the cell when there is an inflammatory insult or intrusion in a cell. The entire
pathway is activated by an orderly response unit, such as B cell activation, cells of innate
immune system are first activated by NF-kB at the site of inflammation, injury or infection
( en- eriah , 2002). NF-kB is a nuclear transcription factor, it has the capability to modify
the cell’s biology by activating or repressing hundreds of target genes and it plays a central
role in the onset of inflammation (Hayden M.S., and Ghosh S., 2012). Thereby, NF-κB is
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directly involved in the regulation as well as the expression of various inflammatory genes
including IL-1β, TNF-α, and COX-2, leading to an activated mitogen-activate protein kinases
(MAPKs) pathway by TNF-α, which can activate all the three groups of MAP kinase
extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38
(Guadalupe Sabio. and Roger J. Davi., 2014).
Decursinol angelate belongs to the genus Angelica L. of the family of Umbellifera, isolated
and extracted from the roots of Angelica gigas Nakai and other plants. The genus Angelica L.
contains more than 60 species (Chen J.T., Yang C.R., 2004). The coumarin compound from
the roots of Angelica gigas has been a subject of comprehensive research because of its
potent therapeutic properties. Shehzad et al has reported that decursin and Decursinol
angelate (A Shehzad et al., 2018) have shown great potential in treating various chronic
inflammatory diseases. Several studies have reported potential role the NF-κ inhibition by
decursin and DA on different cancer cells and macrophage cells (J H. Kim et al., 2006
; A Shehzad et al., 2016; S U. Islam et al., 2018; W J.Kim et al , 2010). The anti-
inflammatory role of decursin derivatives has also been reported in mouse model of asthma
(E J. Yang et al., 2009). Therefore, this study was carried out to comprehend the mechanism
underlying the effects of DA on TPA induced ear inflammation mice model. To the best of
our knowledge, we could not find any literature on in-vivo model of skin inflammation using
DA. In this model, we had topically applied TPA as an inflammagen over a period of 4 days
on mice ears, which induced epidermal hyperplasia and skin inflammation consisting of
erythema, edema and infiltration of plasma and polymorphonuclear leukocyte (PMN) to the
tissues that had undergone disrupted homeostasis. The symptoms of which are similar to
persistent chronic skin inflammation (Lihua Tang., Kepeng Wang., 2016). DA was able to
inhibit the macrophages from polarizing and differentiating in RAW 264.7 cells. We

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observed that administration of DA to mice ears topically resulted in significant inhibition of
TPA-induced ear erythema and edema. DA was also able to reduce the expression of
different pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and enzymes (COX-2, iNOS)
and MCP-1, MIP-2, MIP-1β , MDA from the mice inflamed ears. In addition, we found that
NF- B p65 translocation to the nucleus was blocked, and there was also significant reduction
of TPA-induced MAPK activation. Therefore, DA can be a reliable plant extracted compound
that can be considered as a potential therapeutic drug for the treatment of skin inflammation
related diseases.
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Materials and methods
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2.1. Chemicals
TPA (12-O-tetradecanoylphorbol-13-acetate), DMEM, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), DAPI (4′,6-diamidino-2-phenylindole), dichlorodihydro
-fluorescein diacetate (H2DCFDA), dimethylsulfoxide (DMSO) were purchased from Sigma-
Aldrich, St. Louis, USA. Fetal bovine serum was supplied by Gibco, USA. Primary
antibodies β-actin, p-p38, p-ERK, COX2, n-NOS, i-NOS, e-NOS, TNF-α, NFκB p65 were
purchased from bioworld technology, Inc. All other chemicals and reagents were of the
highest analytical grade available. The details of primary and secondary antibodies used in
the study were given in the supplementary table 1A-B.
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2.2. Synthesis of decursinol angelate
In order to synthesize DA, we prepared the crude extract from the roots of Angelica gigas
(100 grams) as previously described (J Lim et al., 2001). All the reactions were performed
under inert atmosphere (argon or nitrogen) and glassware were oven-dried prior to use.
Briefly, the homogenized root sample of the plant was dissolved in 70% of Ethanol (solvent)
and allowed to boil for 8 hours at 600C. After boiling, the mixture of solvent was filtered and

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extracted. The extraction procedure was performed for 3 times and later we concentrated the
crude sample using rotary evaporator at 400C and recovered the extract using lyophilizer.
Further, to purify decursinol from the crude extract, we dissolved the sample in MeOH/H2O
at a dilution of (1:1), and K2CO3 was added to the crude extract and kept at 100oC for 24h for
reflux. The mixture was then cooled, and EtOAc was added in order to separate the organic
and hydrophilic layers. The organic layer was further concentrated using MgSO4 and the
residue was purified by column chromatography. The total yield of decursinol was 56%,
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melting point: 177-179, Rf = 0.2 (Hexane:EtOAc=1:1). H NMR (CDCl3, 400 MHz) δ 7 51
(d, 1H, J = 9.4 Hz, H4), 7.11 (s, 1H, H5), 6.70 (s, 1H, H10), 6.15 (d, 1H, J = 9.6 Hz, H3), 3.81
(t, 1H, J = 5.2 Hz, H7), 3.04 (dd, 1H, 2J = 16.8 Hz, 3J = 4.8 Hz, H6A), 2.77 (dd, 1H, 2J = 16.8
3
Hz, J = 5.6 Hz, H6B), 1.90 (br. s, OH), 1.33 (s, 3H), 1.29 (s, 3H), 13C NMR (CDCl3, 100
MHz) δ 161 4, 156 5, 154 2, 143 2, 129, 116 5, 113 3, 112 9, 104.8, 78.2, 69.1, 30.7, 25.1,
22.1.
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To further isolate and purify decursinol angelate from decursinol, we mixed 1 gram of
decursinol (4.06 mmol) with 992 mg of (N,N-dimethylamino) pyridine (DMAP, 8.12 mmol),
and 481 mg of (Z)-2-methylbut- 2-enoyl chloride (4.06 mmol) in 30 ml of H2 l2,and kept
in stirring condition for 24 hours at 0℃ under nitrogen. Followed by the dilution of sample
mixture with EtOAc (50 mL) and washed with saturated solution (30 mL), 0.1%
HCl solution (30 mL), and water (30 mL) at the end. The organic layers were dried over
MgSO4, filtered and evaporated under reduced pressure. After removal of solvent, the
resulted residue was purified by column chromatography (n-hexane: EtOAc = 1:1) with silica
gel (230-400 mesh particle size, pore size 60 Å, Merck). The reactions were monitored by
thin-layer chromatography (TLC) on commercial aluminum plates pre-coated with silica gel
60 (F-254, Merck). For further validation, the dried TLC plate was dipped in freshly prepared

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potassium permanganate solution (0.5% KMnO4 in 1 N NaOH) and immediately visualized
under UV light at 254 nm. Purity of the compound was further analyzed by NMR spectra,
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which were obtained using a Bruker-250 spectrometer 250 MHz or 400 MHz for H NMR
and 62.5 MHz or 100 MHz for 13C NMR, reported as parts per million (ppm) from the
internal standard tetramethylsilane (TMS). Chemicals and solvents were of commercial
reagent grade and used without further purification. Elemental analyses were taken on
Hewlett-Packard model 185B elemental analyzer. The yield of decursinol angelate was 82%,
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Melting point 79-80oC (lit.23 78-80oC), Rf = 0.7 (hexanes: EtOAc = 1:1). H NMR (CDCl3,
400 MHz) δ 7 57 (d, 1H, J = 9.2 Hz, H4), 7.14 (s, 1H, H5), 6.81-6.79 (m, 1H, H3’), 6.79 (s,
1H, H10), 6.19 (d, 1H, J = 9.4 Hz, H3), 5.12 (t, 1H, J = 4.8 Hz, H7), 3.28 (ddd, 1H, 2J = 16.8
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Hz, J = 4.8 Hz, J = 1.2 Hz, H6A), 2.92 (dd, 1H, J = 16.8 Hz, J = 5.0 Hz, H6B), 1.77-1.74
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(m, 6H), 1.39 (s, 6H). C NMR (acetone-d6, 100 MHz) δ 167 2, 160 9, 157 3, 155 2, 144 3,
138.5, 130.2, 129.2, 117, 113.9, 113.8, 104.6, 79.2, 77.7, 71, 25.2, 23.5, 14.3, 12.1.
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2.3. DPPH free radical scavenging assay
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The free radical scavenging properties of the DA was determined using 1,1-Diphenyl-2-
picrylhydrazyl (DPPH) assay (Dey D.K et al., 2019). In this assay, ascorbic acid (10 μM) was
considered as a positive control. In 96-well biochemical assay plate, different concentrations
of DA (10, 30, 60 μM) and ascorbic acid were treated with 200 μl of the ethanolic DPPH
solution, followed by incubation in dark for 30 min at room temperature; the absorbance was
then measured at 517 nm by using a spectrophotometer (UV-2120 Optizen, Mecasys, South
Korea).The scavenging ability of the free radical was calculated as follows:
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Scavenging ability %
where At and A0 represent the readings of the sample and blank, respectively, at 517 nm.

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2.4. Cell culture
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Normal human keratinocyte cell (HaCaT) and murine macrophage cell line RAW 264.7 were
cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-
streptomycin on 37oC incubator and facilitated with 5% CO2.
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2.5. MTT assay
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The cytotoxicity of DA on HaCaT cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay. Briefly, cells were cultured in 96 well plates at a
density of 3 x 104 cells/ml and incubated at 37oC humidified and 5% CO2 incubator. After 48
hours when the cells had reached 65-70% confluency the cells were treated with DA at
varying concentrations. After 24 h of incubation, 10 μl of MTT (3-(4,5-demethylthiazol-2-
3yl)-2,5-dephenyltetrazolium bromide, 5mg/ml) reagent was added to each well along with
100 μl of DMEM and allowed to incubate for 3 h at room temperature. The culture medium
containing MTT was discarded carefully and 50 μl of DMSO (dimethyl sulfoxide) was added
to all wells and shaken in the dark for 30 minutes at room temperature for proper dissolution
of the formed formazan products. The optical density for each well was measured at 570 nm
wavelength using a microplate reader (TECAN 200 infinite PRO).
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2.6. Role of DA on macrophage cells
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To evaluate the effect of DA on macrophage cells, we treated murine macrophage RAW
264.7 cells with TPA (10 nM) (C. Wang et al., 2003) prior to DA treatment with different
concentration (10, 30, 60 μM). Further we evaluated the effect of TPA and DA on the cell
density using MTT assay and membrane fluidity using neutral red uptake assay as previously
described (X Li et al , 2012; W Chen et al., 2010, Chauhan AK., 2014). Briefly, RAW 264.7
cells (3 x 104 cells/ml) were seeded into 96 well plates. After 48 hours, cells were treated with

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TPA (10 nM). After 12 hours of incubation TPA pre-treated cells were incubated with various
concentrations of DA (10, 30, 60 μM) and incubated for next 24 hours at 37oC humidified
and 5% CO2 incubator. After the incubation period, the supernatants were discarded and 200
μl of 0.075% neutral red was added to all the wells and further incubated for 30 mins at 37oC.
The neutral red solution was aspirated after 30 mins and the wells were rinsed with 200 μl of
PBS for 3 times with careful precision. The cells in the wells were lysed with 200 μl of 100
mM acetic acid: dehydrated alcohol =1:1 (v/v) and incubated for 8h at 4oC for sufficient
schizo analysis of the cells and for the release of phagotrophic neutral red. After 8h, the
optical density was measured at 490 nm wavelength using a microplate reader (TECAN 200
infinite PRO). The extent of phagocytosis and macrophage activation was expressed through
the OD values.
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2.7. TPA-induced skin inflammation and histology
Male 6–8 weeks old BALB/c mice were purchased from the Orient Bio, Inc. (Seoul, South
Korea). The mice were housed in the animal breeding center of Pohang Technopark and were
kept at room temperature of 20 ± 2°C, relative humidity of 50 ± 5% and light intensity of 12
hours using a constant temperature and humidity device. Prior to starting this study, all
animal-related experiments were conducted after obtaining approval from the Pohang
Technopark Laboratory Ethics Committee (approval number ABCC2018004).
The skin inflammation was induced on the left and right ear of each mouse (n=5) by topical
application of TPA as previously described (H Y. Song et al., 2008; Sun Hwa. Lee et al.,
2012; E H. Ahn et al., 2010). TPA (10 μg) was used as an inflammagen which was dissolved
in 20 μl of acetone and applied to the inner and outer surfaces of the mice ears every 24 h for
4 days. Following 1 h of TPA treatment, DA was applied topically on the ears of the mice.
Next, after 1h of DA treatment to the ears, its thickness was measured using a digital

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thickness gauge (Mitutoyo, Tokyo, Japan). On the final fourth day, we sacrificed the mice
and the whole ear weight was measured and a 8-mm diameter punch was made in each mice
to collect the tissue for the histological analysis.
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2.8. Hematoxylin and Eosin staining
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Ear biopsies were fixed in 4% paraformaldehyde and stored at 4oC for a day. Tissues were
embedded in paraffin and sectioned at a thickness of 8 μm after de-paraffinization and
hydration. The slides were washed with distilled water and stained with hematoxylin for 10
min. Excess amounts of stain were removed with 1% HCl-EtOH solution, followed by
washing with ammonia water and distilled water. Background counterstaining was performed
with eosin, followed by ethanol and xylene washes, cover slip were added to the slides and
was analyzed under a light microscope (both at 20x and 40x resolution).
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2.9. Western blot analysis
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In order to evaluate the protein expression before and after treatment of the mice tissue
sample, we prepared the tissue lysate and loaded them on SDS- polyacrylamide gels. Proteins
were resolved by electrophoresis and were transferred into a polyvinylidene difluoride
membrane (PVDF). The blots were immersed in 5% non-fat milk prepared in PBS and
incubated for 1 h, after which the membranes were probed with the antibody against the
specific protein of interest for 2 hours at 37°C; followed by probing with an appropriate
HRP-conjugated secondary antibody for 1 h at room temperature. All washes between and
after incubations were done by wash buffer; after the final wash, blots were visualized using
enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences
Inc., Piscataway, NJ).
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2.10. Quantitative analysis of cytokines
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Ear tissues were homogenized with a protease inhibitor cocktail (Sigma–Aldrich, St. Louis,
MO, USA) was incubated on ice for 15 min in the presence of 0.1% Triton X-100. The
homogenate was further centrifuged at 15,000×g for 10 min. After centrifugation, TNF-α,
IFN-γ and IL-6, IL-1β, PGE2, MCP-1, MIP-2 in the supernatants were measured by ELISA
(R&D Systems Quantikine ELISA kits) and absorbance measured by TECAN
spectrophotometry. The details of ELISA kits are mentioned in supplementary table 2.

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2.11. Estimation of nitrite level in mice
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The level of nitrite was estimated by using Griess Reagent (Sigma, USA). The ear tissue
homogenized sample was mixed with Griess reagent in a ratio of 1:1, followed by incubation
for 15 min at room temperature. After 1 h, absorbance was recorded at 540 nm using
spectrophotometer (UV-2120 Optizen, Mecasys, South Korea). To determine the amount of
nitrite, a standard curve was plotted using sodium nitrite as per the instructions of the Kit
(G7921, Molecular Probes, life technologies, ThermoFisher, Austria). The standard curve
was used for estimating the concentration of nitrite in each group of the samples.
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2.12. Estimation of Lipid peroxidation in mice
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The level of malondialdehyde (MDA) was measure using Malondialedehyde assay kit
(Abcam) The homogenized ear tissue sample was mixed with 600 μl of thiobarbituric acid
(TBA). The TBA-sample was incubated at 900C for 60 minutes in a hot water bath. After
boiling, the samples were incubated at room temperature for 10-20 mins on an ice bucket.
200 μl of T A-sample reaction mixture was pipetted into a 96 well microplate. The
absorbance was immediately measured at 532 nm wavelength.
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The concentration of MDA was calculated as :
MDA =
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Where, A = Amount of MDA in sample calculated from the standard curve (nmol). mg =
Original tissue amount used (e.g. 10 mg). mL = Original plasma volume used (0.020 mL). 4
= orrection for using 200 μL of the 800 μL Reaction Mix D = Sample dilution factor if
sample is diluted to fit within the standard curve range (prior to reaction well set up).
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2.13. Immunohistochemical staining
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For immunohistochemical staining, de-paraffinized sections of ear biopsy blocks were used.
The de-paraffinized slides were placed in xylene twice (each 5 minutes). The slides were
transferred to 100% ethanol and kept thrice (3 minutes each). After 100% ethanol wash,
slides were transferred to 95% ethanol for 3 minutes. The endogenous peroxidase activity
was blocked by incubating slides for 10-15 minutes in H2O2 in PBS. The slides were than
washed in PBS and kept under blocking buffer (Thermofisher scientific). The slides were

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incubated with primary antibodies (Bioworld Technology) for 3h at room temperature.
After incubation, slides were washed with PBS (3 times) and incubated with secondary
antibody for 30 mins. DAB substrate solution was added to the slides and the slides were
incubated for 15 mins. The slides were rinsed in water and counterstained with haematoxylin
for 30-60 seconds and the dehydration process was carried out after which the slides were
fixed and observed under the microscope (Olympus BX51 Fluorescence Microscope).
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2.14. Immunofluorescence staining
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For immunofluorescence staining, the paraffin-embedded slides were permeabilized in 100%,
70%, 50% and 30% ice-cold methanol for 5 mins each. After ethanol permeabilization, the
slides were washed with Phosphate buffered saline with Tween-20 (PBST) twice for 10
minutes each followed by acetone permeabilization at -20oC for 7 mins. The slides were
washed again with PBST for 10 minutes and incubated with the peroxidase blocking agent
(methanol+ DMSO+ 15% H202) for 10 minutes. The slides were again washed with PBST
and incubated in blocking agent PDT in 3% BSA 30 minutes. After blocking the slides were
incubated in primary antibodies against NF-κB-p65 and TNF-α overnight at 4oC. Next day
the slides were washed 4 times with PDT and incubated in the dark for 2 h with FITC
conjugated secondary antibody and counterstained with DAPI (1µg/ml) for 30 mins. The
slides were washed with PBST and images were collected under the fluorescence microscope
(Olympus BX51 Fluorescence Microscope).
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2.15. Statistical analysis
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The results for all the experiments in this study were expressed as a mean ± standard
deviation (SD) of the experiments, and the statistical significance was calculated using
ANOVA test from prism software; where *represents p-values < 0.05, **represents p-values
< 0.01, and ***represents p-values < 0.001.
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3. Results
3.1. Synthesis and antioxidant potential of DA
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The schematic diagram represented in Fig. 1A shows the isolation process of DA from
Angelica gigas nakai extract. The synthesized compound was further confirmed through
HPLC and NMR analysis. NMR spectrum was very clean and the purity was over 99.5%

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with no bi-product. (Supplementary Figure 1). In our study, DA was further evaluated for the
antioxidant property represented in Fig. 1B. As observed, the percentage inhibition potential
of DA (60 μM) was 50%, with a high statistical significant (p<0.001) and thus, validates the
hypothesis that DA could be beneficial to reduce the inflammation in the host body. The
observed result was similar to the positive control taken in the assay. Ascorbic acid (10 μM)
significantly (p<0.001) scavenged the DPPH free radical from the micro environment.
Therefore, the result demonstrated the antioxidant potential of DA.
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3.2. Cytotoxic assessment of DA on normal human keratinocytes
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To illustrate the potential of DA on the treatment of chronic skin inflammation, first, we
ensured its toxicity for further applications. Therefore, we treated HaCaT cells with different
doses of DA (10, 30, 60 µM), where DMSO (0.1 %) was taken under consideration to
normalize the effect of the solvent. The results from MTT assay for cell viability was
observed to be normal at DMSO 0.1% (98.02%) compared to DA 10, 30 and 60 µM (95.85,
85.71, 84.87 % respectively) having no significant difference (p< 0.1163). Morphological
analysis (Fig. 1D) also revealed the non-cytotoxic behaviour of DA on the normal human
keratinocytes. In addition, it does not show any significant changes in cell viability and
density (Fig. 1D). The results confirmed and satisfied the safety concern for its further
application against TPA induced inflammation in-vitro and in-vivo model.
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3.3. Effect of DA on TPA-induced macrophage activation
To study the potential of DA against TPA induced macrophage activation, we performed
MTT and neutral red uptake assay on RAW-246.7 cells. For MTT assay, the cells were pre-
treated with TPA at 10 nM and then treated with DA at various concentrations (10, 30, 60
μM). To evaluate the morphological changes, we collected cell images after 24 h of treatment
(Fig. 2A). The results of MTT assay shows a significant (p<0.0001) activation of macrophage
as observed on TPA treated group which was subsided after DA (60 μM) treatment (Fig. 2B).
As one of the important factors in the uptake of particles by phagocytic cells is the membrane
fluidity, so we also analysed the specific function of murine macrophages by neutral red
uptake assay. The cells were treated with TPA (10 nM) and DA at varying concentrations
(10, 30, 60 μM) After the addition of neutral red solution DA at 60 μM showed the
significant (p< 0.01) activity in reducing the TPA induced macrophage activation giving an
optical density of 0.017 in comparison with TPA (0.082, p < 0.001) treatment (Fig. 2C). The

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result was also further evaluated on mice ear tissues. Immunofluorescence staining of
intercellular adhesion molecule (ICAM-1), which is highly expressed during macrophages
activation (S F. Bernatchez et al.,1997) revealed that TPA treatment group had exaggerated
overexpression of macrophages and infiltratory cells as compared with control and DA
treatment groups, (Fig. 6A) analysed through the uptake of more green fluorescence stain on
TPA group. We also evaluated the levels of monocyte chemoattractant protein-1 (MCP-1)
and macrophage inflammatory protein 2 (MIP-2) from mice ear tissue homogenates. It was
found that TPA treatment group for both MCP-1 and MIP-2 had a higher expression
concentration (404 pg/ml and 488.5 pg/ml) which were significantly lowered upon DA
treatment for both DA 10 and 25 mg/kg ( 210, 171.5 pg/ml, p < 0.0001) and (162.65, 130.5
pg/ml, p < 0.0001 ). Western blotting (Fig. 4C) analysis for macrophage inflammatory
protein1β (MIP-1β) also reduced in a dose dependent manner compared to TPA groups (p<
0.0001) indicating the role of DA in subduing Inflammatory cell infiltration triggered by TPA
treatment.
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3.4. Anti-inflammatory potential of DA on TPA-induced ear inflammation in mice ears
TPA has shown the potential to activate the macrophages in order to induce the inflammation
(P L. Stanley et al., 1991; H Y. Ha et al., 2006). Therefore, we designed the in-vivo
experiment to induced mice ear edema using TPA as a model for acute and chronic
inflammation (Fig. 3A). Topical application of TPA (10 μg/ear) on BALB/c mice ear over a
period of 4 days was shown to stimulate a skin inflammatory response consisting of erythema
and edema (Fig. 3B). TPA treated group aggravated the swelling of ears, increased ear
thickness and ear weight (p<0.01) (Fig.3 C, D). Topical application of DA, (10 and 25
mg/kg) after 1h of TPA application once a day for 4 days ameliorated TPA induced ear
edema and decreased the number of inflammatory cells in a dose dependent manner. The
result was also further analysed through H&E staining (Fig. 4A), where TPA treated group
showed increased re-epithelialization, epidermal hyperplasia, accumulation of granulation
tissue, newly formed blood vessels and infiltration of inflammatory cells. There was also
increased number of inflammatory cells infiltration at the TPA treatment regions of the ear
and an increase in epidermal thickness (Fig. 4B). We observed a significant (p<0.001)
reduction of epidermal hyperplasia, inflammatory cells and epidermal thickness in the DA
(10 and 25 mg/kg) treated groups indicating an effective anti-inflammatory potential of DA
on in-vivo model system.

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3.5. Effects of DA on NF-κB and MAP kinase pathway on TPA-induced inflammation in mice
ears
In order to trigger a pro-inflammatory cytokine response by immune cells, NF-κ and MAPK
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are the fundamental pathways that are needed to be activated (P Zhang et al., 2005; T
Lawrence., 2009). Therefore, we topically applied TPA on the inner and outer regions of the
mice ears for 4 days. After 1 hour of TPA treatment, DA at a dose of 10 and 25 mg/kg was
administered topically on the TPA treated region of mice ears. From the ear tissue
homogenates, we performed a western blotting analysis of MAP kinase pathway markers
such as p-ERK (p< 0.0041) and p-p38 (p< 0.0004) protein kinase. The phosphorylated ERK
and p38 marker was markedly downregulated in a dose dependent manner on DA treatment
groups, in comparison to TPA treatment only mice group (Fig. 4C). TPA also induced the
activation of NF-κB, which is a well-known redox-sensitive transcription factor regulating
the expression of many pro-inflammatory genes (T Liu et al., 2017; Dev Bhatt., Sankar
Ghosh., 2014). NF-κB p65 subunit expression was also downregulated in a dose-dependent
manner in DA treatment groups observed through western blotting of IKKα (p< 0.0002),
IKKγ (p< 0 0019), Iκ α (p< 0.0001) and Fκ -p65 (p< 0.0007) (Fig. 4C). Further, we also
assessed the transcriptional targets of NF-κB in the tissue sample by analysing their
expression and comparing with the expression of control, TPA and DA treatment groups.
Accordingly, we observed that the expressions of TNF-α (p< 0.0049), e-NOS (p< 0.0178), i-
NOS (p< 0.0114), n-NOS (p< 0.0012), and COX2 (p<0.0028) (Fig. 4C) were significantly
higher in the TPA treated group when compared to the control group. We also observed a
significant dose dependent decrease in their protein levels after treatment with DA.
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3.6. Effect of DA on the generation of reactive oxygen species
ROS is known to react directly with the double bonds of polyunsaturated fatty acids
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(PUFAs), leading to the production of lipid hydroxides which thereby contribute to the

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disruption of necessary bio-molecules required for biological mechanisms (G Barrera., 2012).
As per the western blot analysis, we observed DA can significantly reduce the expression of
NOS proteins, so we further evaluated the potential of DA to inhibit oxidative stress induced
by TPA which generates more reactive oxygen species (A Q.Khan et al., 2013). The mice ear
tissue homogenates were analyzed for the production malondialdehyde (MDA), which is a
major marker of oxidative stress (Draper H., Hadley M., 1990). We observed that DA groups
10 and 25 mg/kg (1.7 and 1.2 nmol/mg) was able to significantly reduce the production of
MDA as compared with TPA group (2.8 nmol/mg) with a significant difference of (p<
0.0050) (Fig.5B). The ear biopsy were also stained and analyzed for the uptake of green
fluorescent dye H2DCFDA (A Sengupta et al., 2010). As illustrated in Fig. 5A, the intensity
of green fluorescence markedly decreased in a dose-dependent manner in DA treated groups
(10 and 25 mg/kg). Whereas, TPA treatment only group had an intense green fluorescence
stain indicating a higher production of ROS. The results revealed a reduced level of reactive
oxygen species generation after topical application of DA. We further performed NO assay,
as depicted in Fig. 6B, TPA treated group showed significantly higher production of NO
(43.05 μM) as compared to control group (7.13 μM). Topical application of DA (10-25
mg/kg) significantly reduced NO production on TPA challenged groups (22.93 μM and 14.21
μM,) and a significant difference of (p< 0.0006) respectively. Concurrently, western blotting
analysis of antioxidant enzymes (Fig. 6C), catalase (p< 0.0012), superoxide dismutase (SOD-
1) (p< 0.0001) and manganese superoxide dismutase (SOD-2) (p< 0.0006) were also
increased significantly on DA treatment groups. Collectively, the result of the experiment
also supports the finding of Fig. 1B.
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3.5. Effect of DA on the production of pro-inflammatory cytokines in TPA induced ear
inflammation

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In order to further validate the results of western blotting analysis, we performed ELISA and
immunohistochemistry and immunofluorescence staining. The results of ELISA estimation
are presented in Fig. 6 (C, D, E, F, G). TPA treatment aggravated the mice ears resulting in
the over expression of pro-inflammatory cytokines, whereas, 10 and 25 mg/kg treatment of
DA significantly reduced their production rate as observed. The level of TNF-α, IL-6, IL-1β,
PGE2, and IFN-γ was recorded lesser in the control group (18 38, 12 82, 369 56, 62 26 and
15.79 pg/ml) respectively. However, their level was found to be elevated in TPA induced
inflammatory group (141.63 pg/ml, p<0.001; 558.01 pg/ml, p<0.001; 369.56 pg/ml,
p<0.001; 62.26 pg/ml, p<0.001; and 37.93 pg/ml; p<0.001) respectively. But after treatment
with DA of 10 mg/kg and 25 mg/kg their level of significantly reduced (36.19 pg/ml,
p<0.001; 57.67 pg/ml, p<0.001; 157.57 pg/ml, p<0.001; 42.69 pg/ml, p<0.05; and 25.8
pg/ml, p<0.05) and (18.24 pg/ml, p<0.001; 43.16 pg/ml, p<0.001; 129.66 pg/ml, p<0.001;
and 29.15 pg/ml, p<0.01; and 20.80 pg/ml, p<0.01) respectively. Altogether, Topical
application of DA (25 mg/kg) was found to be potent enough to inhibit the level of PGE2, e-
NOS, i-NOS, n-NOS and COX-2 in TPA- induced ear inflammation model system.
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The results were also validated by immunohistochemistry analysis. As observed, the mice ear
biopsy had revealed lesser accumulation of NF-κB-p65, TNF-α, and IL-1β on DA (10 and 25
mg/kg) in comparison with the TPA treated group, which had higher expression of these pro-
inflammatory cytokines determined through brown reaction products (Fig. 7 A, B, C).
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In particular, to determine the impact of DA on NF-κB-p65, TNF-α expression,
immunofluorescent staining was also performed. As shown in Fig. 8, the fluorescent intensity
of both NF-κB-p65 (Fig. 8A) and TNF-α (Fig. 8B) were highly upregulated in TPA induced
inflammatory model when compared to its respective control sample. Conversely, topical
treatment of DA reduced the fluorescent intensity of NF-κB-p65 and TNF-α, providing the
evidence of its inhibition capacity against the TPA induced ear inflammation. Taken together,
this result indicates that DA could be a good therapeutic compound for different kinds of
inflammatory diseases.
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4. Discussion
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Angelica gigas is a medicinal plant of Korean origin, having high amounts of DA as
compared to other Dangui of Chinese and Japanese origin. We synthesized DA from the roots
of Angelica gigas and performed HPLC and NMR analysis to confirm the structure and

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purity of DA (Supplementary Figure 1-A, B, C, D, E, F). Inflammation-inducer of phorbol
ester origin such as TPA induces a similar type of chronic inflammatory symptoms and is
also rooted in the development of different types of cancers (Tobias Bald et al., 2016). After
Topical application of TPA over a period of 4 days on mice ears, we observed a rush of
inflammatory cell infiltration, comprising of the influx of polymorphonuclear leukocytes
(neutrophils) on the site of inflammation which are primed by cytokines and mobilized by
chemokines and the secretion of a various different pro-inflammatory cytokines and MHC
class II molecules triggers the activation of T cells (Wright HL., 2010). The activated T-
helper cells play a central role in the subsequent invasion of monocytes, which differentiate
into tissue macrophages (Jinfang Zhu., 2008). The inflammatory reaction induced by TPA
and different irritants can be quantified by measuring the ear weigh and ear thickness,
appropriate for preliminary screening of anti-inflammatory drugs ( He et al., 2013). We
further investigated the molecular and cellular mechanisms of inflammation after TPA
application on mice ears and performed histological, ELISA and western blotting analysis.
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Our in-vivo experiment was able to substantiate the previously published papers of DA on an
in-vitro anti-inflammatory model system. DA at both doses (10-25 mg/kg) was able to
significantly inhibit TPA-induced ear inflammation, and subsequently suppress the activation
of macrophages and infiltrating inflammatory cells. To further justify our experiment, we
investigated the markers of macrophages and inflammatory cells such as ICAM-1, MIP-2,
MCP-1, MIP-1β The involvement of ROS and its role in various skin inflammatory diseases
have been extensively studied (Bickers DR., Athar M., 2006). So, we tested the potential role
of DA to inhibit oxidative stress induced by application of TPA. The nitric oxide synthase
enzymes, which catalyse the synthesis of nitric oxide, were significantly downregulated
resulting in reduced free radical NO production upon DA treatment. MDA, which is one of
the final products of polyunsaturated fatty acid peroxidation, overproduced by excessive free
radicals in the cells was also significantly downregulated. DA administration was also able to
enhance the activity antioxidant enzymes such as catalase, SOD-1, and SOD-2. The
inactivation of NF-κ could be validated by the reduction of F-κ p65 subunit and the
down-regulation of NF-κ target genes such as T F-α, IL-6 which was established through
ELISA, staining and western blotting analysis, overall, leading to the repression of pro-
inflammatory enzymes and cytokines. Conclusively, we suggest DA can be a good
therapeutic compound for different kinds of inflammatory reactions on the skin and

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inflammation-related diseases. However, further more experiments would be required to
validate the compound as a potent drug for clinical trials
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Conflict of interest
The authors declare no competing financial interest.
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Acknowledgements:
This research was financially supported by the Ministry SMEs and Startups(MSS), Korea,
under the “Regional Specialized Industry Development Program ( o P0004934)” supervised
by the Korea Institute for Advancement of Technology (KIAT) and "This work was
supported by a grant from the Nakdonggang National Institute of Biological Resources
(NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea
( NNIBR201902105 )."
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Reference
Adeeb Shehzad., Salman Ul. Islam., Eun Mi. Ahn., You Mie. Lee., and Young Sup. Lee.
443
444
445
446
447
448
449
450
451
452
453
454
(2016). Decursinol angelate inhibits PGE2-induced survival of the human leukemia HL-
60 cell line via regulation of the EP2 receptor and NF kB pathway. CANCER BIOLOGY
& THERAPY. VOL. 17, NO. 9, 985–993.
Barrera, G. (2012). Oxidative stress and lipid peroxidation products in cancer progression
and therapy. Oncol. 2012–137289.
Ben Neriah, Y. (2002). Regulatory functions of ubiquitination in the immune system. Nat.
Immunol. 3, 20–26.
Bickers DR., Athar M. (2006). Oxidative stress in the pathogenesis of skin disease. J
Invest Dermatol; 126:2565-75.
Cathy Wang., James H. Steer., David A. Joyce., Kirk Hm. YIP., Ming H. Zheng., Jiake
Xu. (2003). 12-O-tetradecanoylphorbol-13-acetate (TPA) Inhibits Osteoclastogenesis by
Suppressing RANKL-Induced NF-κ Activation J Bone Miner Res. 18(12):2159-68.

ACCEPTED MANUSCRIPT
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
Chauhan AK., Jakhar R., Paul S., Kang SC. (2014) Potentiation of macrophage activity
by thymol through augmenting phagocytosis. Int Immunopharmacol. 18(2):340-6.
Chen J.T., Yang C.R. (2004). Researches in Angelica L. Nat Prod Res Dev 16(4).359-
365.
Shehzad A., Parveen S., Qureshi M., ., Lee Y.S. (2018). Decursin and decursinol
angelate: molecular mechanism and therapeutic potential in inflammatory diseases.
Inflammation Research . 67:209–218
Degan D., Ornello R., Tiseo C., Carolei A., Sacco S., Pistoia F. (2018). The Role of
Inflammation in Neurological Disorders. Curr Pharm Des. 24(14):1485-1501.
Dev Bhatt., and Sankar Ghosh. (2014). Regulation of the NF-κ -Mediated Transcription
of Inflammatory Genes. Front Immunol. 5: 71.
Dey D.K., Khan I. , Kang S.C. (2019). Anti-bacterial susceptibility profiling of Weissella
confusa DD_A7 against the multidrug-resistant ESBL-positive E. coli. Microb Pathog.
128:119-130.
Draper H., Hadley M. (1990). Malondialdehyde determination as index of lipid
peroxidation. Methods Enzymol. 186:421-31.
E J. Yang., G Y. Song., J S. Lee., C Y. Yun., I S. Kim. (2009). A novel (S)-(+)- decursin
derivative, (S)-(+)-3-(3,4-dihydroxy-phenyl)-acrylic acid 2,2-dimethyl-8-oxo-3,4-
dihydro-2H,8H-pyrano[3,2-g]chromen-3- yl-ester, inhibits ovalbumin-induced lung
inflammation in a mousemodel of asthma, Biol. Pharm. Bull. 32 444–449.
Eun Hee. Ahn., Dae Won. Kim., Hye Won. Kang., Min Jae. Shin., Moo Ho. Won., Joon
Kim., Dong Joon. Kim., Oh Shin. Kwon., Tae Cheon. Kang., Kyu Hyung. Han., Jinseu
Park.,Won Sik. Eum., Soo Young. Choi. (2010). Transduced PEP-1-ribosomal protein S3
(rpS3) ameliorates 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice.
Toxicology. 276 192–197.

ACCEPTED MANUSCRIPT
480
481
482
Firestein G. S. (2003). Evolving concepts of rheumatoid arthritis. Nature 423, 356-361.
Guadalupe Sabio. and Roger J. Davis. (2014). TNF and MAP kinase signaling pathways.
Semin Immunol. 26(3): 237–245.
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
Ha H.Y., Kim Y., Ryoo Z.Y., Kim T.Y. (2006). Inhibition of the TPA induced cutaneous
inflammation and hyperplasia by ECSOD. Biochem Biophys Res Commun. 348:450–8.
Hayden M.S., and Ghosh S. (2012). NF-kB, the first quarter-century: Remarkable
progress and outstanding questions. Genes Dev. 26, 203–234.
J. MacMicking., Q.W. Xie., C. Nathan. (1997). Nitric oxide and macrophage
function,Annu. Rev. Immunol. 15 323–350.
Jinfang Zhu.,William E. Paul. (2008). CD4 T cells: fates, functions, and faults.
Blood; 112:1557-1569
Jongdoo Lim., AIk Hwan. Kim., Hyeon Ho. Kim., Kyung Seop. Ahnc., and Hogyu Hana.
(2001). Enantioselective syntheses of decursinol angelate and decursin. Tetrahedron
Letters 42. 4001–4003.
Jung Hee. Kim., Ji Hye. Jeong. , Sung Tak. Jeon., Ho Kim., Jiyeon Ock., Kyoungho Suk.
,Sang In. Kim., Kyung Sik. Song., and Won ha. Lee. (2006). Decursin Inhibits Induction
of Inflammatory Mediators by Blocking Nuclear Factor- κ activation in Macrophages.
Mol Pharmacol .69:1783–1790.
Khan A.Q., Khan R., Qamar W., Lateef A., Rehman M.U., Tahir M., Ali F., Hamiza
O.O., Hasan S.K., Sultana S. (2013). Geraniol attenuates 12-O-tetradecanoylphorbol-13-
acetate (TPA)-induced oxidative stress and inflammation in mouse skin: possible role of
p38 MAP Kinase and NF-κ Exp Mol Pathol. 94(3):419-29.

ACCEPTED MANUSCRIPT
502
503
504
505
506
507
508
509
510
Korhonen, R., Lathi, A., Kankaanranta H., and Moilanen E. (2005). Nitric oxide
production and signaling in inflammation. Curr. Drug Targets Inflamm. Allergy 4, 471-
479.
Libby P., Ridker P. M., and Maseri A. (2000). Inflammation and atherosclerosis.
Circulation. 105, 1135-1143.
Li Q., Verma I.M. (2002). NF- B regulation in the immune system. Nat Rev
Immunol.2:725–734.
Mosser D.M., Edwards J.P. (2008). Exploring the full spectrum of macrophage activation.
Nat Rev Immunol.; 8:958–969.
511
512
513
514
515
516
517
518
519
520
Nathan C., Ding A.( 2010). Non resolving inflammation. Cell.; 140:871–882
Ping Zhang., Michael Martin., Suzanne M. Michalek., and Jannet Katz. (2005). Role of
Mitogen-Activated Protein Kinases and NF-κ in the Regulation of proinflammatory and
anti-Inflammatory Cytokines by Porphyromonas gingivalis Hemagglutinin B. Infect
Immun. 73(7): 3990–3998.
Qian Zhang., Michael J. Lenardo., and David Baltimore. (2017). 30 years of NF-κ : a
blossoming of relevance to human pathobiology. Cell.168(1-2): 37–57.
Rainer H., Straub and Carsten Schradin. (2016). Chronic inflammatory systemic diseases:
An evolutionary trade-off between acutely beneficial but chronically harmful programs.
Evolution, Medicine, and Public Health. pp. 37–51.
521
522
523
524
Salman Ul. Islam., Jung Ho. Lee., Adeeb Shehzad. , Eun-Mi. Ahn., You Mie. Lee., and
Young Sup. Lee. (2018). Decursinol Angelate Inhibits LPS-Induced Macrophage
Polarization through Modulation of the Fκ and MAPK Signaling Pathways
Molecules. 23, 1880

ACCEPTED MANUSCRIPT
525
526
527
528
529
Sengupta A., Lichti U.F., Carlson B.A., Ryscavage A.O., Gladyshev V.N. (2010).
Selenoproteins Are Essential for Proper Keratinocyte Function and Skin Development.
PLoS ONE. 5(8): e12249.
Sergei I. Grivennikov., Florian R. Greten, and Michael Karin. Immunity, Inflammation,
and Cancer. (2010) Cell 140, 883–899.
530
531
532
533
534
535
536
537
538
539
540
541
Song H.Y., Lee J.A., Ju S.M., Yoo K.Y., Won M. H., Kwon H.J., Eum W.S., Jang S.H.,
Choi S.Y., Park J. (2008). Topical transduction of superoxide dismutase mediated by
HIV-1 Tat protein transduction domain ameliorates 12-O-tetradecanoylphorbol-13-
acetate (TPA)-induced inflammation in mice. Biochem. Pharmacol. 75, 1348–1357.
Stanley P.L., Steiner S., Havens M., Tramposch K.M. (1991). Mouse skin inflammation
induced by multiple topical applications of 12-O-tetradecanoylphorbol-13-acetate. Skin
Pharmacol 4:262–71.
Stephanie F. Bernatchez., Matthew R. Atkinson., Patrick J. Parks. (1997). Expression
molecule-l of intercellular adhesion on macrophages in vitro a marker of activation.
Biomaterials. 18 1371-1378.
Sullivan G.W., Sarembock I.J., Linden J.(2000). The role of inflammation in vascular
diseases.J Leukoc Biol. 67(5):591-602.
542
543
544
545
546
547
548
Sun Hwa. Lee., Dae Won. Kim., Seon Ae. Eom., Se Young. Jun., Meeyoung Park., Duk
Soo. Kim., Hyung Joo. Kwon., Hyeok Yil. Kwon., Kyu Hyung. Han., Jinseu Park., Hyun
Sook. Hwang., Won Sik. Eum., Soo Young Choi. (2012). Suppression of 12-O-
tetradecanoylphorbol-13-acetate(TPA)-induced skin inflammation in mice by transduced
Tat-Annexin protein. BMB Rep. 45(6):354-9.
Ting Liu., Lingyun Zhang., Donghyun Joo.,and Shao Cong. Sun. (2017). NF-κ signaling
in inflammation. Signal Transduct Target Ther. 2: 17023.

ACCEPTED MANUSCRIPT
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
Tobias Bald., Jennifer Landsberg., Philipp Jansen., Evelyn Gaffal., Thomas Tuting.
(2016). Phorbol ester-induced neutrophilic inflammatory responses selectively promote
metastatic spread of melanoma in a TLR4-dependent manner. ONCOIMMUNOLOGY,
VOL. 5, NO. 2, e1078964
Toby Lawrence. (2009). The Nuclear Factor NF-κ Pathway in Inflammation Cold
Spring Harb Perspect Biol. 1(6): a001651.
W hen , W Zhang , W Shen , and K Wang (2010) “Effects of the acid polysaccharide
fraction isolated from a cultivated Cordycepssinensis on macrophages in vitro” Cellular
immunology. vol. 262, no. 1, pp. 69–74.
Won Jung. Kim., Min Young. Lee., Jung Hee. Kim., Kyoungho Suk., Won Ha. Lee.
(2010). Decursinol angelate blocks transmigration and inflammatory activation of cancer
cells through inhibition of PI3K, ERK and NF-κB activation. Cancer Letters 296 35–42.
Lihua Tang.,and Kepeng Wang. (2016).Chronic Inflammation in Skin Malignancies .J
Mol Signal. 11: 2. pp.ꢀ1–13.
Wright HL., Moots RJ., Bucknall RC., Edwards SW. (2010). Neutrophil function in
inflammation and inflammatory diseases. Rheumatology; 49: 1618-31.
X Li., Y. Su., J Sun., and Y. Yang. (2012). Chicken embryo extracts enhance spleen
lymphocyte and peritoneal macrophages function Journal of Ethnopharmacology, vol.
144, no. 2,pp. 255–260.
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Figure Legends
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Figure 1. DA exhibits non toxicity on HaCaT cells and Free radical scavenging potential
(A) Preparation of Decursinol angelate from Angelica gigas extract (B) Free radical
scavenging activity of DA on DPPH (C) MTT assay to evaluate the cytotoxicity of DA on
normal human keratinocyte cells (HaCaT) (D) Structural morphology of HaCat cells after

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Treatment of DA at different concentration doses (10, 30, 60 μM) and DMSO (0.1%) control
(no treatment). Scale bar (100 μm). The data are represented as the means ± S.D. of three
independent experiments *p< 0.05, **p<0.01, ***p< 0.001 ***p<0.00. Statistical
significance was analysed by prism ANOVA.
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Figure 2. DA reduces TPA induced macrophage activation on Mice ear biopsy and
RAW 264.7 cells (A) Morphological analysis of Raw 264.7 cells 24 hours after treatment
with different doses of DA was compared with the TPA treatment (10 nM) (B) cell density
was analyzed by MTT assay at 72 hours. (C) The effect of TPA on membrane fluidity of
murine macrophage cell line was validated and its reduction was validated by dose dependent
effect of DA using Neutral Red uptake assay. The data are represented as the means ± S.D. of
three independent experiments *p< 0.05, **p<0.01, ***p< 0.001, ***p<0.001 and ns (non-
significant). TPA vs control, DA 10 mg/kg and 25 mg/kg vs TPA. Statistical significance was
analysed by prism ANOVA.
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Figure 3. DA Inhibits TPA induced epidermal hyperplasia and ear edema (A) Schematic
representation of the experimental procedure. (B) Images of mice ear (n=5) collected every
day after TPA and DA treatment over a period of 4 days. (C) Inhibition of TPA induced
epidermal hyperplasia and ear edema was analyzed by measuring the following parameters.
Ear thickness was measure every day after topical treatment of TPA and DA for 4 days (D)
Ear weight of different mice group after sacrifice.
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Figure 4. DA ameliorates TPA induced epidermal hyperplasia and inhibits MAPK and
NFκB pathway (A) H&E staining images of ear biopsy tissue. Black arrow : The regions
in the black arrow indicates epidermal hyperplasia and re-epithelialization with newly formed,
thick epidermis that was complete as observed on TPA alone group. Square: The regions
under the square indicate massive aggregation of granulation tissue granulation tissue. It also

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occurs in all wounds during the healing process and it also occurs in chronic inflammation. It
comprises of fibroblasts surrounded by abundant ECM, newly formed blood vessels,
scattered macrophages, and some other inflammatory cells. (B) Measurement of epidermal
thickness of different groups in μm. Scale bar (100 μm). (C) DA significantly reduced the
inflammatory response challenged by TPA (10 μg/ear), which was validated through western
blotting analysis. We observed a significant downregulation of MIP-1β (p< 0.0001), TNF-α
(p< 0.0049), e-NOS (p< 0.0178), i-NOS (p< 0.0114), n-NOS (p< 0.0012), COX-2 (p<
0.0028), p-ERK(p< 0.0041), p-p38 (p< 0.0004), IKKα (p< 0.0002), IKKγ (p< 0.0019), IκBα
(p< 0.0001), NFκB p65 (p< 0.0007) at DA 10 and 25 mg/kg treatment groups compared with
TPA group. Densitometry analysis of the respective proteins was evaluated by Image J
software, and results were normalized by β-actin. The data are represented as mean ± S.D. of
three independent experiments *p< 0.05, **p<0.01, ***p < 0.001 and ns ( non-significant).
TPA vs control, DA 10 mg/kg and 25 mg/kg vs TPA.
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Figure 5. DA inhibits ROS production and promotes antioxidant activity (A)
Immunofluorescent detection of DCFH-DA on mice ear biopsy section. DCFH-DA is stained
green and nucleus is stained blue with DAPI. Measurement of ROS by H2DCFDA showed
reduction of free radicals on DA (10-25 mg/kg) group as compared with TPA group. TPA
alone group is shown to have more intense green fluorescence stain indicating more
production of ROS, while the intensity of green fluorescence dye decreased on DA groups.
Magnification= 20x. (B) Ear tissue samples were evaluated for Malondialdehyde production.
(C) Western blotting analysis of antioxidant enzyme markers, Catalase (p< 0.0012), SOD-1
(p< 0.0001), SOD-2 (p< 0.0006). Densitometry analysis of the respective proteins was
evaluated by Image J software, and results were normalized by β-actin. The data are
represented as the means ± S.D. of two independent experiments *p< 0.05, **p<0.01, ***p <

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0.001 and ns (non-significant). TPA vs control, DA 10 mg/kg and 25 mg/kg vs TPA.
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Figure 6. DA suppresses TPA induced macrophage activation and inhibits production
of different pro-inflammatory cytokines (A) Macrophage activation was analysed through
ICAM-1 overexpression on mice ear biopsies. Immunofluorescent detection of ICAM-1 for
macrophage polarization and activation. ICAM-1 is stained in green and nucleus is stained
blue with DAPI. The experiment was performed to validate the activation of macrophage
leading to highly expressed ICAM-1 on TPA treatment group which was analysed by the
uptake of more green fluorescence stain on the ear biopsy tissue sections as compared with
control (no treatment) and DA (10-25 mg/kg) group (n= 5 per group) Magnification= 20x.
(B-I) Measurement of inflammatory cytokines from the ear biopsy tissues. Inhibitory effect
of DA on TPA-induced inflammatory cytokines from the ear biopsy tissues in mice ears.
Mice ears (n=5) were treated with TPA (10 μg/ear) once a day for 4 days. DA (10 and 25
mg/kg) was topically applied to mice ears 1 h after TPA treatment. Mice ear extracts were
prepared and analysed for pro- inflammatory cytokine expression. Supernatant fractions of
homogenates from ear biopsies were examined for cytokine production using ELISA and NO
assay, IL-6, TNF-α IL-1β, PGE2, IFN-γ, M P-1, MIP-2. The p values for TPA was compared
with control and p value for sample DA 10 mg/kg and 25 mg/kg was compared with TPA.
The data are represented as the means ± S.D. of three independent experiments *p< 0.05,
**p< 0.01, ***p< 0.001, ***p< 0.001 and ns (non-significant). TPA vs control, DA 10
mg/kg and 25 mg/kg vs TPA. Statistical significance was analysed by prism ANOVA.
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Figure 7. Immunohistochemical staining of TPA treated ear biopsies after formalin
fixation and paraffin-embedded sections (A) NF-κ p65 ( ) T F-α ( ) IL-1β TPA group
had more concentration of NF-κ p65 as compared to DA (10-25 mg/kg) as determined
through browning reaction observed in the stained tissue sections. Scale bar (100 μm).

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Magnification= 20x.
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Figure 8. Immunofluorescence staining of TPA treated ear biopsies after formalin
fixation and paraffin-embedded sections (A) Immunofluorescent detection of TNF-α.
TNF-α is stained in green and nucleus is stained blue with DAPI. (B) Immunofluorescent
detection of NF-к p65 F-к p65 is stained in green and nucleus is stained blue with
DAPI. The experiment was conducted in order to validate the inhibition of TNF-α and NF-
κ -p65 expression by DA (10-25 mg/kg) as compared with TPA only group which was
analysed by the uptake of more green fluorescence stain on the ear biopsy tissue sections. (n=
5 per group) Magnification= 20x.
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Graphical Abstract: Role of DA in TPA-induced ear inflammation.
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Supplementary Figure 1: (A) Chromatogram illustrating the identification of different
compounds present in the root extract of Angelica gigas. (B) The total yield of DA from
decursinol. (C) The pictorial diagram representing the HPLC condition applied in order to
extract the pure compound. (D-E) The figures representing the standard curve and the
chromatogram of the purified compounds respectively. (F) NMR analysis was performed to
confirm the compound purity and its structure.
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Supplementary Figure 2: Original western blotting images used in Figure 4.
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Supplementary Figure 3: Original western blotting images used in Figure 5.
Supplementary table 1A: List of primary antibodies used in the study.
Supplementary table 1B: List of secondary antibodies used in the study.
Supplementary table 2: List of ELISA assay kits used in the study

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