Synthesis of eugenol derivatives and its anti-inflammatory activity against skin inflammation

Article abstract of DOI:10.1080/14786419.2018.1528585

Eugenol is a phytochemical present in aromatic plants has generated considerable interest in the pharmaceutical industries mainly in cosmetics. A series of eugenol esters (ST1-ST7) and chloro eugenol (ST8) have been synthesized. The structures of newly synthesized compounds were confirmed by ¹H and ¹³C NMR and mass spectrometry. In an effort to evaluate the pharmacological activity of eugenol derivatives, we explored its anti-inflammatory potential against skin inflammation using in-vitro and in-vivo bioassay. Synthesized derivatives significantly inhibited the production of pro-inflammatory cytokines against LPS-induced inflammation in macrophages. Among all derivatives, ST8 [Chloroeugenol (6-chloro, 2-methoxy-4-(prop-2-en-1-yl)-phenol)] exhibited most potent anti-inflammatory activity without any cytotoxic effect. We have further evaluated the efficacy and safety in in-vivo condition. ST8 exhibited significant anti-inflammatory activity against TPA-induced skin inflammation without any skin irritation effect on experimental animals. These findings suggested that ST8 may be a useful therapeutic candidate for the treatment of skin inflammation.

Full text of DOI:10.1080/14786419.2018.1528585

Natural Product Research
Formerly Natural Product Letters
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Synthesis of eugenol derivatives and its anti-
inflammatory activity against skin inflammation
Anil Kumar Maurya, Karishma Agarwal, Amit Chand Gupta, Archana Saxena,
Zulfa Nooreen, Sudeep Tandon, Ateeque Ahmad & Dnyaneshwar Umrao
Bawankule
To cite this article: Anil Kumar Maurya, Karishma Agarwal, Amit Chand Gupta, Archana Saxena,
Zulfa Nooreen, Sudeep Tandon, Ateeque Ahmad & Dnyaneshwar Umrao Bawankule (2018):
Synthesis of eugenol derivatives and its anti-inflammatory activity against skin inflammation,
Natural Product Research, DOI: 10.1080/14786419.2018.1528585
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1528585
Synthesis of eugenol derivatives and its anti-
inflammatory activity against skin inflammation
Anil Kumar Mauryaa,b, Karishma Agarwalc,d, Amit Chand Guptaa, Archana
Saxenaa, Zulfa Nooreenc, Sudeep Tandonc,d, Ateeque Ahmadc,d and
Dnyaneshwar Umrao Bawankulea,d
aMolecular Bioprospection Department, CSIR-Central Institute of Medicinal and Aromatic Plants,
b
c
Lucknow, India; Sai Nath University, Ranchi, India; Process Chemistry and Technology, CSIR-Central
d
Institute of Medicinal and Aromatic Plants, Lucknow, India; Academy of Scientific and Innovative
Research, New Delhi 110025, India
ABSTRACT
ARTICLE HISTORY
Received 19 July 2018
Accepted 22 September 2018
Eugenol is a phytochemical present in aromatic plants has gener-
ated considerable interest in the pharmaceutical industries mainly
in cosmetics. A series of eugenol esters (ST1-ST7) and chloro
eugenol (ST8) have been synthesized. The structures of newly syn-
thesized compounds were confirmed by 1H and 13C NMR and
mass spectrometry. In an effort to evaluate the pharmacological
activity of eugenol derivatives, we explored its anti-inflammatory
potential against skin inflammation using in-vitro and in-vivo bio-
assay. Synthesized derivatives significantly inhibited the production
of pro-inflammatory cytokines against LPS-induced inflammation in
macrophages. Among all derivatives, ST8 [Chloroeugenol (6-chloro,
2-methoxy-4-(prop-2-en-1-yl)-phenol)] exhibited most potent anti-
inflammatory activity without any cytotoxic effect. We have further
evaluated the efficacy and safety in in-vivo condition. ST8 exhib-
ited significant anti-inflammatory activity against TPA-induced skin
inflammation without any skin irritation effect on experimental ani-
mals. These findings suggested that ST8 may be a useful thera-
peutic candidate for the treatment of skin inflammation.
KEYWORDS
Eugenol; skin; macrophage;
inflammation; mice
CONTACT Ateeque Ahmad
a.ahmad@cimap.res.in; D. U. Bawankule
du.bawankule@cimap.res.in
Supplementary Data for this article can be accessed here at https://doi.org/10.1080/14786419.2018.1528585
Anil Kumar Maurya and Karishma Agarwal contributed equally to this study.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
A. KUMAR MAURYA ET AL.
1. Introduction
Essential oils and its constituents have received special attention as a source of poten-
tially useful bioactive compounds in pharmaceutical and cosmeceutical industries due
to their pharmacological properties (Polzin et al. 2007). Eugenol, 2-methoxy-4-(2-pro-
penyl) phenol is a phytochemical obtained from Syzigium aromaticum, Ocimum
Sanctum and Perry (Daniel et al. 2009), widely known as clove it is native to tropical
Asia (Polzin et al. 2007) and cultivated in different regions of the world (Agra et al.
2008). Eugenol is used in folk medicine for its biological properties, such as antimicro-
bial, antifungal, antioxidant (Chainy et al. 2000; Zhang et al. 2017), anesthetic,
antiseptic (Daniel et al. 2009), anticarcinogenic (Zheng et al. 1992), anti-nociceptive,
^
anti-inflammatory activities (Fonseca et al. 2016), antiviral, and antiallergic (Kim et al.
2001) effects. It is also used in the food industry as a flavoring agent (Hatami et al.
2010). Eugenol derivatives synthesized by esterification reactions in the hydroxyl group
with different carboxylic acids as well as semi-synthetic derivatives of eugenol through
catalytic oxychlorination has shown potential antibacterial activities (da Silva et al.
2018; Pinheiro et al. 2018). Eugenol isolated from aromatic crops has been reported
for promising pharmacological activities and this has prompted us to synthesize its
derivatives to investigate its therapeutic effect. In the present study, we have synthe-
sized the eugenol esters (ST1-ST7) and chloro eugenol (ST8) to examine its therapeutic
effect on inflammatory response with special emphasis in skin inflammation using in-
vitro and in-vivo bioassays. Skin is well known for its functional role as a protective
physical barrier and dynamic organ that has some other recognized functions, such
as endogenous homeostasis, metabolism, and sensory input. In addition, skin actively
participates in Immunological regulatory processes and inflammatory responses (Bos
1997). Inflammation is a key molecular mechanism which includes skin disorders
(Kong and Xu 2018), inflammatory skin diseases have a significant impact on the
quality of life of human being and represent an enormous financial trouble.
However, some inflammatory or immunological reactions lead to chronic inflamma-
tion processes, such as psoriasis or to intolerable skin inflammation conditions, such
as contact dermatitis, which requires medication (Rauh et al. 2011). During inflamma-
tion, the inflammatory region is infiltrated with mononuclear cells such as mono-
cytes, macrophages and lymphocytes, producing a wide range of Inflammatory
mediators, such as interleukin-1 beta (IL-1b), interleukin-6 (IL-6), tumor necrosis fac-
tor-alpha (TNF-a) and cyclooxygenase-2 (COX-2) play important roles in inflammatory
diseases (Libby et al. 2002; Bertolini et al. 2004). Atopic dermatitis (AD) is a chronic
or chronically relapsing, pruritic inflammatory skin disease (Kim et al. 2018). Topical
application of TPA has been used to screen for topically applied anti-inflammatory
steroids and non-steroid agents and events of the inflammatory processes such as
edema, cell infiltration, and proliferation. Our results demonstrated that, among all
synthesized derivatives, ST8 [Chloroeugenol (6-chloro, 2-methoxy-4-(prop-2-en-1-yl)-
phenol)] exhibited most potent anti-inflammatory activity without any cytotoxic
effect in the in-vitro study. We have further evaluated the efficacy and safety in in-
vivo condition. ST8 exhibited significant anti-inflammatory activity against TPA-
induced skin inflammation without any skin irritation effect on experimental animals.

NATURAL PRODUCT RESEARCH
3
2. Results and discussion
2.1. Chemistry
An ester is a chemical compound derived from an acid or its chloride (organic or inor-
ganic) in which at least one –OH (hydroxyl) group is replaced by an –O–alkyl (alkoxy)
group. Esters of eugenol derivatives were prepared from respective acid chlorides reagent
such as acetyl, propinyl, valeryl, isovaleryl, palmitoyl, lauryl, benzoyl in presence of pyridine
and sulphuric acid at room temperature or heating as per preparation required of differ-
ent derivatives. Isolation of eugenol (EUG) and its synthetic approach of all eugenol deriv-
atives such as eugenyl valerate (ST1), eugenyl isovalerate (ST2), eugenyl palmitate (ST3),
eugenyl propionate (ST4), eugenyl acetate (ST5), eugenyl laurate (ST6), eugenyl benzoate
(ST7) and chloroeugenol (ST8) are well written in experimental section of the paper.
2.2. Pharmacology
All the synthesized eugenol derivatives at 0.01, 0.1 and 1% concentration were evaluated
for their anti-inflammatory status against the production of pro-inflammatory cytokines
(TNF- a, IL-6) using ELISA technique in LPS-induced inflammation in macrophage cells.
Production of pro-inflammatory cytokines was significantly (p < 0.05) increased in vehicle-
treated LPS-stimulated cells when compared with normal un-stimulated cells. All the deriv-
atives exhibited inhibition of pro-inflammatory cytokines production when compared with
vehicle-treated LPS-stimulated cells (Supplementary file Table S1). Among all derivatives,
ST8 (Chloroeugenol) possessed most potent derivatives by significant inhibition of TNF-a,
and IL-6 without any cytotoxic effect as assessed by MTT assay. Several previous studies
reported that molecules derived from essential oil reduce the production of inflammatory
mediators by inhibiting the activation of the inflammatory signaling pathway (Maurya et al.
2014). These pro-inflammatory cytokines are the mediators of various chronic inflammation
linked diseases including skin inflammation (Maurya et al. 2018). To substantiate the
physiological function of ST8, a most potent derivative; we have further evaluated the
therapeutic efficacy and safety profile in-vivo system using TPA-induced skin inflammation
and primary skin irritation study in mice and rabbits respectively. Topical application of ST8
on TPA-induced skin inflammation in the ear of mice exhibited the significant reduction in
ear thickness, edema, ear weight (Figure 1) as well as pro-inflammatory biomarkers in ear
tissue homogenate (Figure 2) in a dose-dependent manner. Experimental evidence has
shown that exposure of skin to 12-O-tetradecanoyl phorbol-13-acetate (TPA) induces a
pleiotropic tissue response encompassing a strong inflammatory reaction similar to that
observed in skin diseases (Kondo et al. 2000). Our observations are similar to the previous
findings that essential oil can reduce the severity of skin inflammation (Boukhatem et al.
2013; Blaskovic et al. 2014). Edema and increased skin thickening are the indicative parame-
ters of increased vascular permeability, edema and swelling within the dermis, and prolifer-
ation of the epidermal keratinocytes during skin inflammation process (De Vry et al. 2005).
It is widely recognized that the secretions of cytokines by keratinocytes in response to
injury are key mediators of the cutaneous inflammatory response (Murphy et al. 2000).
Primary skin irritation study in rabbits revealed that ST8 is safe for topical application on
skin. Previous reports also concluded that essential oils from aromatic plants are not

4
A. KUMAR MAURYA ET AL.
Figure 1. Dose-response effect of ST8 on TPA-induced inflammatory ear swelling in BALB/c mice
ꢀ
(A) Change in ear thickness (B) Ear weight. Results are Mean SEM; n ¼ 6; P < 0.05 (Vehicle
vs Treatment).
Figure 2. Effect of ST8 on inflammatory mediators on TPA-induced ear inflammation in BALB/c
mice (A) Ear homogenate TNF-a and (B) Ear homogenate IL-6. Data are expressed as Mean SEM;
ꢀ
n ¼ 4; P < 0.05 (Vehicle vs Treatment).
irritating to mammalian skin and safe for topical application (Yadav et al. 2013; Liu et al.
2015). Skin Irritation test was conducted to determine the primary skin irritation on rabbit
skin. 72 h after the application of ST8 on rabbit skin. Significant erythema and edema for-
mation were not observed in ST8 treatment site compared to the vehicle-treated site.
Pigmentation, blood oozing, and rough skin were also not observed in both the control
and treatment site. According to Federal Hazardous Substances Act (FHSA) regulations, a
material with a PII of less than 5.00 is generally not considered a primary irritant to the skin.
The PII result concluded that the application of ST8 is not irritant to the rabbit skin.
3. Experimental section
3.1. Chemistry
3.1.1. Distillation & isolation of eugenol
Eugenol was isolated from the essential oil of Ocimum sanctum variety CIM-Ayu having
eugenol content of about 54-60%. 200 kg of fresh herb of the plant was steam

NATURAL PRODUCT RESEARCH
5
7
H3CO
3
8
H3CO
2
4
10
9
H3C(H2C)3OCO
5
1
HO
6
ST1
EUG
H3CO
H3CO
H3C(H2C)14OCO
(H3C)2HCH2COCO
ST 2
ST3
H3CO
H3CO
H3CH2COCO
H3COCO
ST4
ST5
H3CO
H3CO
H3C(H2C)10OCO
O
ST6
O
ST7
H3CO
HO
Cl
ST8
Figure 3. Structure of eugenol derivatives.
distilled in a boiler operated steam distillation unit to obtain 750 ml of essential oil.
The oil was treated with anhydrous sodium sulphate to remove any moisture in the
oil. 500 ml of the oil was then fractionated in a packed fractionating column of 40
mm dia x 1200 mm height packed with hyflux wire mesh packing. Fractionation was
carried out under the vacuum of 5-8 mm Hg. Fractionation data is given in Table S2.
Four major fractions were collected in which 253 ml eugenol of 97.2% purity was
obtained which was further chromatographed over silica gel using hexane as the elut-
ing solvent to achieve 99% purity of eugenol. The purity of eugenol was confirmed by
GC. The isolated eugenol was stored under sub-zero temperatures till further
experiments.
3.1.1.1. Synthesis of esters and chloro eugenol. Structure of esters and chloro deriv-
atives of eugenol are depicted in Figure 3 and spectroscopic data and spectral figures
1
of H and 13C NMR are given in the Supplementary file.
Eugenyl valerate (ST1): To a solution of eugenol 10 g (60.97 mmol), valeryl chlor-
ide 25 g (150.33 mmol) and 1 ml pyridine added was left at room temperature for
overnight and then refluxed over the water bath for 1 hr. The reaction mixture was
cooled to room temperature, diluted with water and extracted with chloroform (3 x 25
ml). The organic layer was washed with water, dried over sodium sulphate and evapo-
rated to dryness. The crude reaction product was purified by a silica gel column and
eluted with hexane/chloroform as a liquid (purity 97.5%); Yield 72%.
Eugenyl isovalerate (ST2): In a round bottom flask, 10 gm (60.97 mmol) of
eugenol was dissolved in chloroform. To it isovaleryl chloride 25 gm of (150.33 mmol)

6
A. KUMAR MAURYA ET AL.
and 5 ml pyridine added. The reaction mixture was refluxed over a water bath for 2
hr. The progress of the reaction was monitored using TLC in hexane ethyl acetate.
After the completion of the reaction, the reaction mixture was worked up using water
and chloroform. The organic layer was then separated, dried over anhyd. sodium sul-
phate and concentrated till dryness. The crude reaction product was purified by silica
gel column and eluted with hexane/ethyl acetate as a liquid (purity 96.5%),
Yield 79.0%.
Eugenyl palmitate (ST3): Eugenol 10 g (60.97 mmol), palmitoyl chloride 25 g
(90.90 mmol) and 5 ml pyridine was taken in a round bottom flask and refluxed for 1
hr. The reaction mixture was cooled to room temperature, diluted with water and
extracted with chloroform (3 x 25 ml). The organic layer was washed with water, dried
over sodium sulphate and evaporated to dryness. The crude reaction product was
purified by silica gel column and eluted with hexane/ethyl acetate as a liquid (purity
98.5%); Yield (69%), Liquid.
Eugenyl propionate (ST4): 10 g (60.97 mmol) of eugenol was taken in a round
bottom flask, 10 g (108.08 mmol) of propinyl chloride was added to the solution of
Eugenol. In this mixture, 10 ml of pyridine was added dropwise. After the addition of
pyridine the reaction mixture began to solidoify. The reaction mixture was left at
room temperature for 2 hrs. After completion of the reaction, Reaction mixture was
diluted with water and extracted with chloroform (3 x 25 ml). Organic layer was
washed with water, dried over sodium sulphate and evaporated to dryness. The crude
reaction product was purified by silica gel column and eluted with hexane/ethyl acet-
ate as a liquid (purity 98.5%); Yield 78%.
Eugenyl acetate (ST5): To a solution of Eugenol 5 g (30.48 mmol), acetyl chloride
12.5 g (126.58 mmol) and 2.5ml of sulphuric acid were added. The reaction mixture
was then refluxed for 2-3 hr. the progress of the reaction was monitored using TLC.
After the completion of the reaction, the reaction mixture was cooled to room tem-
perature, diluted with water and extracted with chloroform (3 x 25 ml). The organic
layer was washed with water, dried over sodium sulphate and evaporated to dryness.
The crude reaction product was purified by a silica gel column and eluted with hex-
ane/ethyl acetate as a solid (purity 98.1%); Yield 79%.
Eugenyl laurate (ST6): Eugenol 5 g (30.48 mmol), lauryl chloride 15 g (73.17
mmol) and 2.5ml of sulphuric acid was taken in a Round bottom flask and refluxed for
2-3 hrs. After the completion of the reaction, the reaction mixture was cooled to room
temperature, diluted with water and extracted with chloroform (3 x 25 ml). The
organic layer was washed with water, dried over sodium sulphate and evaporated to
dryness. The crude reaction product was purified by a silica gel column and eluted
with hexane/ethyl acetate as a liquid (purity 97.3%); Yield 74%.
Eugenyl benzoate (ST7): To a solution of eugenol 5 g (30.48 mmol), benzoyl chlor-
ide 15 g (60.14 mmol) was added. To this reaction mixture 2.5ml of sulphuric acid was
added dropwise and the reaction mixture was refluxed for 2- 3 hrs. After the comple-
tion of the reaction, the reaction mixture was worked up using Water and Chloroform.
The crude reaction product was purified using a column chromatography in a silica
gel (60-120 mesh) which yielded a pure solid compound in hexane/ethyl acetate;
Yield 71%.

NATURAL PRODUCT RESEARCH
7
Chloroeugenol (ST8): In a round bottom flask, 10 g (60.97 mmol) of eugenol was
dissolved in 20 ml of carbon tetrachloride. 8 g (59.25 mmol) of sulphuryl chloride was
added to this reaction mixture dropwise. The reaction mixture was left at room tem-
perature for 11-12 hrs and the progress of the reaction was monitored using TLC.
After the completion of the reaction, the reaction mixture was diluted with water and
extracted with chloroform (3 x 25 ml). The organic layer was washed with water, dried
over sodium sulphate and concentrated under reduced pressure. The crude reaction
product was purified by a silica gel column and eluted with hexane/ethyl acetate as a
liquid (purity 96.5%); Yield 78%.
3.2. Pharmacology
3.2.1. In-vitro study
The macrophage cells were isolated from the peritoneal cavities of female Swiss albino
mice after an intra-peritoneal injection of 1.0 mL of 1% peptone (BD Biosciences, USA)
3 days before harvesting. Culture macrophage cells were pre-treated with 0.01, 0.1
and 1% of synthesized derivatives of eugenol (ST1-8). The cells were stimulated with
LPS (0.1mg/mL) for 24 h and collected supernatants were immediately frozen at ꢁ80ꢂC.
Pro-inflammatory mediators (TNF-a, and IL-6) were quantified from the supernatant
using ELISA method according to the manufacturer’s instructions (BD Biosciences,
USA). The values of TNF-a and IL-6 were expressed as pg/mL. ST8, a most potent lead
were further evaluated check its effect on cytotoxicity using 3-(4, 5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium (MTT) assay.
3.2.2. In-vivo study
BALB/c Mice (25–35 g) and New Zealand white rabbits were used for efficacy and tox-
icity study, respectively. Experiments were performed according to the ethical guide-
lines suggested by the Institutional Animal Ethics Committee (IAEC) and Committee
for the Purpose of Control and Supervision of Experiments on Animals, Government
of India.
After 1 week of adaptation, the mice were randomly divided into five groups of
four mice each. Topical inflammation was induced in the experimental mice except for
the normal group of mice by the topical application of TPA (2.5lg). ST8 was dissolved
in acetone and administered topically for 30 minutes before application of TPA.
Acetone alone was applied as the vehicle to normal and vehicle-treated mice group.
The experimental animals were divided as follow
Group 1: Normal control
Group 2: Vehicle control þ TPA
Group 3: ST8 (0.01%) þ TPA
Group 4: ST8 (0.1%) þ TPA
Group 5: ST8 (1%) þ TPA

8
A. KUMAR MAURYA ET AL.
TPA-induced skin inflammation was maximum at 6 hours. The area and degree of
ear inflammation were assessed in order to calculate a macroscopic score of ear
inflammation. A macroscopic score between 0 and 7 was attributed to all mice ears.
Score 0-1 meant no skin inflammation. Skin inflammation was scored between 2-3
when very slight, 4-5 when moderate, and 6-7 when severe as described by Maurya
et al. 2018 (Supplementary file; Figures S3 and S4). Edema was expressed as the
increase in ear thickness due to the inflammatory challenge. Ear thickness was meas-
ured before and after the induction of the inflammatory response by using an elec-
tronic digital micrometer (Aerospace Instruments).
At 6 hours, when TPA-induced inflammation was maximal, animals were euthanized
by ether asphyxiation and 1 cm diameter punch of ear tissue wet weight was taken
for quantification of inflammatory mediators from ear tissue homogenate. Production
of pro-inflammatory cytokines (TNF-a and IL-6) were quantified from tissue homogen-
ate using commercially available mouse specific enzyme immune assay (EIA) kits (BD
Biosciences, USA) as per the manufactures instruction. To study the skin toxicity effect,
primary skin irritation study was performed for the topical application of ST8 in four
healthy rabbits (Observations were made at 1, 4, 24, 48 and 72 h to assess individual
erythema and edema. The primary irritation index (PII) was determined using the fol-
lowing formula; PII = Test site score – Control site score.
4. Statistical Analysis
Data were expressed as Mean SEM. For statistical analysis, one-way ANOVA followed
by Tukeys test was used. Probability (P) values less than 0.05 were considered
significant.
5. Conclusion
The results of the study demonstrated that topical application of eugenol and its
derivatives are effective as an anti-inflammatory agent against TPA-induced skin
inflammation and among all derivatives, ST8 [Chloroeugenol (6-chloro, 2-methoxy-4-
(prop-2-en-1-yl)-phenol)] was most potent. This study suggests the suitability of
eugenol and its derivatives in skin care formulations for pharmaceutical purposes with
special reference to skin inflammation.
Acknowledgments
The authors are grateful to Director of the institute for kind support during the progress of
the work.
Disclosure statement
The authors declare that there is no conflict of interests regarding the publication of this paper.

NATURAL PRODUCT RESEARCH
9
Funding
The research was supported by Council of Scientific and Industrial Research (CSIR), New Delhi,
India for financial support through BSC-0203 and HCP-007.
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