Anti-inflammatory activity of Jatropha curcas Linn. latex in cream formulation on CD68 expression in mice skin wound

Abstract

Aim: The purpose of the present study was to determine the potential of Jatropha curcas latex in the cream formulation on CD68 immune expression (macrophages) during inflammatory phase wound healing process in mice skin.

Materials and Methods: Amount of 12 two-months-old male mice were used between 30 and 40 g. To surgical procedures, wound skin incision was performed 2.0 cm in length until subcutaneous on the paravertebral of each animal. The treatment was carried under locally anesthetized with procaine cream. The mice were allotted into four groups of each, entire surface of each group wound covered by base cream control, sulfadiazine 0.1% cream, J. curcas latex cream 10% and, 15%, respectively. All experiments were performed twice a day for 3 days. The wound healing was assayed in stained histological sections in immunohistochemical of the wounds. CD68 expression was investigated under a microscope.

Results: The results showed that the cream from the 10% and 15% latex of J. curcas revealed moderate immune reaction to CD68 on wound healing.

Conclusion: We concluded that the latex cream of J. curcas possesses anti-inflammatory activity in wound healing process of mice skin.

Full text

Veterinary World, EISSN: 2231-0916 99
Veterinary World, EISSN: 2231-0916
Available at www.veterinaryworld.org/Vol.11/February-2018/1.pdf
RESEARCH ARTICLE
Open Access
Anti-inflammatory activity of Jatropha curcas Linn. latex in cream
formulation on CD68 expression in mice skin wound
Muhammad Nur Salim1, Dian Masyitha2, Abdul Harris3, Ummu Balqis1, Cut Dahlia Iskandar2, Muhammad Hambal4
and Darmawi5,6
1. Laboratory of Pathology, Faculty of Veterinary Medicine, Syiah Kuala University, Banda Aceh 23111,
Indonesia; 2. Laboratory of Histology, Faculty of Veterinary Medicine, Syiah Kuala University, Banda Aceh 23111,
Indonesia; 3. Laboratory of Pharmacology, Faculty of Veterinary Medicine, Syiah Kuala University, Banda
Aceh 23111, Indonesia; 4. Laboratory of Parasitology, Faculty of Veterinary Medicine, Syiah Kuala University, Banda
Aceh 23111, Indonesia; 5. Laboratory of Microbiology, Faculty of Veterinary Medicine, Syiah Kuala University,
Banda Aceh 23111, Indonesia; 6. Laboratory of Research, Faculty of Veterinary Medicine, Syiah Kuala University,
Darussalam, Banda Aceh 23111, Indonesia.
Corresponding author: Darmawi, e-mail: darmawi@unsyiah.ac.id
Co-authors: MNS: mnursalim@unsyiah.ac.id, DM: dianchairida@gmail.com, AH: harris57abdul@gmail.com,
UB: ummu.balqis@unsyiah.ac.id, CDI: acut.ifan@gmail.com, MH: hambal.m@unsyiah.ac.id
Received: 12-09-2017, Accepted: 19-12-2017, Published online: 02-02-2018
doi: 10.14202/vetworld.2018.99-103 How to cite this article: Salim MN, Masyitha D, Harris A, Balqis U, Iskandar CD,
Hambal M, Darmawi (2018) Anti-inflammatory activity of Jatropha curcas Linn. latex in cream formulation on CD68
expression in mice skin wound, Veterinary World, 11(2): 99-103.
Abstract
Aim: The purpose of the present study was to determine the potential of Jatropha curcas latex in the cream formulation on
CD68 immune expression (macrophages) during inflammatory phase wound healing process in mice skin.
Materials and Methods: Amount of 12 two-months-old male mice were used between 30 and 40 g. To surgical procedures,
wound skin incision was performed 2.0 cm in length until subcutaneous on the paravertebral of each animal. The treatment
was carried under locally anesthetized with procaine cream. The mice were allotted into four groups of each, entire surface
of each group wound covered by base cream control, sulfadiazine 0.1% cream, J. curcas latex cream 10% and, 15%,
respectively. All experiments were performed twice a day for 3 days. The wound healing was assayed in stained histological
sections in immunohistochemical of the wounds. CD68 expression was investigated under a microscope.
Results: The results showed that the cream from the 10% and 15% latex of J. curcas revealed moderate immune reaction
to CD68 on wound healing.
Conclusion: We concluded that the latex cream of J. curcas possesses anti-inflammatory activity in wound healing process
of mice skin.
Keywords: anti-inflammatory, CD68, Jatropha curcas latex cream, wound healing.
Introduction
On the basis of the physiology of wound heal-
ing, the wounds can be classified as chronic and acute
wounds. Balqis et al. [1] documented that chronic
wound appeared in an 18-years-old male Elephas
maximus sumatranus due to surgical debridement
to remove tumor extraskeletal fibrosarcoma in the
captive elephant. Acute wounds caused by acciden-
tal tripping have been found in wild animals such
as tiger, Panthera tigris sumatrae [2]. Indeed, these
wounds contribute to substantial morbidities such
as increased risk for infection, limb amputation,
and animal death. It is understood that there are
certain essential drugs present in modern medicine
that help in the healing of wounds. There are sev-
eral growth factors which are having the potential
of improving the healing of wounds. Furthermore,
a number of antibiotic compounds have been used
in the treatment of the bacterial infected wounds.
Unfortunately, there is a serious untoward effect such
as carcinogenesis can be generated by some of these
growth factors [3,4]. Using commercial antibiotic
compounds leads to the risk of widespread devel-
opment of resistant bacteria to most of the current
antibiotics. A number of antibiotics resistant can be
demonstrated in Staphylococcus aureus bacteria iso-
lated from a human in Mongolia [5], from milk in
South Africa [6], and from poultry in Malaysia [7].
Multidrug resistance occurred in Escherichia coli
strain [8] and Salmonella spp. [9]. Due to the risks,
various authors made a new path to introduce alter-
native wound healing agents from natural origin.
The process of wound healing occurs in three phases
such as inflammatory, proliferative, and maturation
[10]. Importantly, the inflammatory phase is naturally
intended to remove devitalized tissue and prevent
invasive infection [11]. The macrophages are promi-
nent inflammatory cells in wounds. Macrophages are
phagocytes, contributing to both innate immunity and
cell-mediated immunity. Their function is to stimulate
Copyright: Salim, et al. Open Access. This article is distributed under
the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Commons
license, and indicate if changes were made. The Creative Commons
Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.

Veterinary World, EISSN: 2231-0916 100
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lymphocytes and other immune cells to respond to
the pathogens and to phagocytize cellular debris and
pathogens [12,13]. The CD68 protein belongs to a
family of the lyso somal glycoprotein which is spe-
cifically expressed by tissue macrophages including
Langerhans cells. Macrophages have many functions
in wound healing, including host defense, the promo-
tion and resolution of inflammation, the removal of
apoptotic cells, and support of cell proliferation and
tissue restoration following injury [14].
Due to its availability, small size rodent, easy
of handling and low cost, mice are used as suitable
animals model for the study of skin wound heal-
ing that closely parallels to the healing of human
wounds. Various authors choose mice to carry out
their study. For wound healing and antimicrobial
properties, a study employed by Agra et al. [15]
showed that the aqueous extract from Bowdichia
virgilioides stem barks can be used in mice. Jiao
et al. [16] described that the function of cell-medi-
ated immunity of mice was influenced by flavonoid
extracted from stem and leaf of Astragalus mem-
branaceus. Uche and Aprioku [17] demonstrated
that the analgesic and anti-inflammatory effects
of methanol extract of Jatropha curcas leaves in
mice. Previously, Mujumdar and Misar [18] used
the mice as animals’ model in the study of the
anti-inflammatory activity of J. curcas roots. In
another study using leaf extract; Salim et al. [19]
noticed that the efficacy of J. curcas Linn. on the
process of wound healing in mice.
J. curcas Linn. from the Euphorbiaceae family
has been used in many parts of the world for various
medicinal purposes [20]. The leaf and latex extracts
of J. curcas contained appreciable amounts of sec-
ondary metabolic compounds [21-23]. Leaves of this
plant reported possessing antivirus on human immu-
nodeficiency virus activity [24]. Extracts from this
plant have been reported to have remarkable anti-in-
flammatory and antibacterial [25], cosmetic [26] and
wound healing [27,28] hemostatic [29], antioxidant,
and anticancer [30] potential. Unfortunately, the
effect of this medication during the inflammatory
phase of wounds healing process has not been fully
defined.The present study was, therefore, conducted
to evaluate J. curcas latex activity in cream formu-
lation on CD68 immune expression (macrophages)
during the inflammatory phase of wound healing pro-
cess in mice skin.
Materials and Methods
Ethical approval
All experimental animal procedures were per-
formed in compliance with the regulation of Animal
Ethics Committee. This research was approved by the
Animal Ethics Committee of Faculty of Veterinary
Medicine, Syiah Kuala University, Banda Aceh,
Indonesia (Approval No. 004/KEPH-C/VII/2017).
Experimental animals
Amount of 12 male mice (Mus muscu-
lus) 2 months old and weighing 30-40 g obtained
from Faculty of Veterinary Medicine, Syiah Kuala
University, Banda Aceh, Indonesia, were used in cur-
rent study. The mice were housed in individual cages
which include drinking water and rations. The mice
were fed a standard laboratory diet and given ad libi-
tum access to food and water. The animals were kept
for acclimatization for 2 weeks.
Preparation of cream
The latex of J. curcas was obtained from a local
farm in Darussalam village, Banda Aceh City, Aceh
Province, Indonesia. The latex was collected in the
morning with a break of leaf stems, latex at capacity
into a sterile bottle. J. curcas latex cream made with
a base of oil in water (O/W) according to methods
Muntiaha et al. [31]. A cream base was added little
by little with the 10% and 15% latex of J. curcas in a
porcelain dish containing 100 g of cream and stirred
until homogeneous at room temperature.
Surgical procedures
All mice were anesthetized locally with procaine
cream, and one wound skin incision was performed
2.0 cm in length until subcutaneous on the paraver-
tebral of each animal. The animals were handled in
accordance with aseptic principles to avoid exogenous
bacterial contamination.
Experimental design
Mice were divided into four groups with three
mice of each. Group 1(A) as a negative control, the
entire surface of wound covered by base cream.
Group 2(B) as a positive control, the entire surface
of wound covered by sulfadiazine 0.1% cream both
Group 3(C) and Group 4(D) as treatment groups.
The C and D, the entire surface of wound covered by
J. curcas latex cream 10% and 15%, respectively. The
wound was treated twice a day at 08.00 am and 18.00
pm until day 3.
Histopathological and immunohistochemical expres-
sion of CD68
Wound skin tissue samples were collected in
10% buffered formalin for histopathological exam-
ination [32,33]. The tissues were processed by rou-
tine paraffin embedding technique, and 5 µm sections
were stained with immunohistochemical staining by
standard methods as described by Darmawi et al. [34],
using streptavidin-biotin complex (SB). For macro-
phages immunoreaction, we used the CD68 mono-
clonal mouse antibody (Dako, Carpentaria, CA) at
dilution 1:100 in PBS. Immunohistochemical label-
ing for each antibody was graded on scale of 0-3
Grades according to the following assessment: No
detectable labeling (Grade 0), weak or mild labeling
(Grade 1), moderate labeling (Grade 2), marked label-
ing (Grade 3) with local, and/or widespread reactivity
as described by Caffo et al. [35].

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Results
The results of the present study showed that the
cream from the 10% and 15% latex of J. curcas revealed
moderate immune reaction to CD68 on wound healing.
The CD68 positive cells were especially detected in
perivascular area. The CD68 monoclonal mouse anti-
body had a strong reaction with antigen on the con-
nective tissue macrophages as seen in Figure-1. On
day 3 wound healing process mice skin, the expres-
sion of CD68 positively reacts in four group treatment.
Immunohistochemical examination with SB staining
revealed macrophages in all the organs examined. The
expression of macrophages in the skin tissue can be
identified by the expression of the CD68 marker for
a positive result. CD68 positive cells closed to peri-
vascular area. Macrophages were identified as brown
color against a pale ground background. Here, we
regarded them as Langerhans macrophages.
Our result showed that the mice wound skins
covered by base cream had a mild immune reaction to
CD68 expression. We found that dramatic change of
mice wounds skins in the appearance of CD68 expres-
sion treated with sulfadiazine 0.1%. We explained that
dramatic changes of mice wound skins were observed
in most of treated with J. curcas latex cream groups.
Comparatively, the concentration of J. curcas latex
cream 10% showed less effect than J. curcas latex
cream 15%. Treatment of mice wounds skins with
J. curcas latex cream 15% resulted in dramatic change
in moderate immune reaction to CD68 expression as
seen in Figure-1.
Discussion
The simplest interpretation of our finding is that
at least some macrophages go through the wound
skins. In the study of Guo et al. [36] showed that
immunohistochemical examination CD68 mac-
rophages can determine in the process of wounds
healing are more specific than histopathological with
routine staining. Macrophages on the tissue can be
identified by marker human CD68. Nucera et al. [37]
described that CD68 was a 110kDa transmembrane
glycoprotein by human monocytes and macrophages.
CD68 can be used for identifying a population of cells
being of mononuclear phagocyte origin, assessing the
number of macrophages infiltrating a wound healing
area. CD 68 expressed by Langerhans cells constitute,
the most abundant macrophage population in the skin
was observed as a major gateway for wound infection.
In another study showed that the majority of macro-
phages found in the perivascular area of wound heal-
ing [28]. In this study, J. curcas latex cream 15% is
an optimum dose that could fasten the inflammatory
phase wound healing process. It was found that the
process of wound healing in D is better than the other
groups.
The present study clearly demonstrated that the
CD68 expression on mice wound skins it can be seen
that the J. curcas latex cream 15% have potential as an
anti-inflammatory. In support of this hypothesis, many
previous reports exist about the effect of using J. cur-
cas as therapeutic agents that contributes to impaired
wound healing process. These extracts showed good
antioxidant [22], coagulant and anticoagulant [38],
analgesic, antibacterial, and anti-inflammatory [25].
A similar outcome was observed the effect of n-hex-
ane J. curcas leaf extract showing the hemostatic
effect that exhibited a significant decreased in bleed-
ing time [29]. The latex from branches has been used
in wound healing, refractory ulcers, and septic gums
and as a styptic in cuts and bruises [28]. These find-
ings are not different from those reported the effect
of J. curcas extract revealed that reduction of bleed-
ing time increased proportionally with the increase in
extract concentration [29]. Regarding the activity of
J. curcas extract to impaired wound healing process
similar for and support those of Shetty et al. [27], who
observed in albino rats showed that extract of J. cur-
cas bark was potential in accelerating wound healing.
There are some secondary metabolic compounds
extracted from J. curcas involved in wound healing
process. Abdelgadir and Staden [20] described that the
leaf and latex extracts of J. curcas contained appreciable
amounts of phenolic and saponin compounds that are
responsible for antimicrobial and antioxidant activities.
Importantly, Esimone et al. [28] explained that flavonoid
quercetin and rutin can improve wound healing process
in the initial phase, namely, the regulation of the expres-
sion of vascular endothelial growth factor (VEGF) for
the growth of new blood vessel and formation of col-
lagen Type III. However, flavonoid compounds play a
role in the early phase of wound healing (inflammatory
phase) to increase the activity of the immune system
of the body such as interleukin-2, proliferation of lym-
phocytes, and macrophages [16]. Moreover, sesquit-
erpenoids are responsible for antimicrobial and anal-
gesic effects, meanwhile, proteins such as curcain are
Figure-1: Photomicrograph of wounds skin on day 3
of treatment (streptavidin-biotin 40×). CD68 positive
cells each group that marked the brown color (arrow).
(a) Minimal immunoreactivity CD68 positive cells on
Group A (Grade 1). (b-d) Moderate immunoreactivity CD68
positive cells in Groups B-D (Grade 2).
a
c
b
d

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responsible for wound healing [20]. Therefore, Jatropha
oil has anti-inflammatory activity [17,18,39]. The leaves
and latex of J. curcas contained phenolic compounds
and saponins that have antioxidant and anti-inflam-
matory activity [40]. Flavonoid as an antioxidant can
reduce free radicals and will bond with free radical that
damage cell membranes [41]. Flavonoids accumulated
in the latex of J. curcas play an essential role in inflam-
matory phase by increasing interleukin-2, proliferation
of lymphocytes, and macrophages [16]. Indeed, sapo-
nins can increase the proliferation of monocytes which
eventually will increase the number of macrophages
that secrete the growth factor which crucial for wound
healing process [42].
The evidence described here argues that the
J. curcas latex cream is also possibly involved in the
early phase of wound healing. In confirmation of our
previous study, we found that J. curcas latex cream
histopathologically could be decreased inflamma-
tory cells infiltration [19]. Various authors noticed
that the purpose of inflammation was interesting to
plasma protein and phagocytic cells to the wound
surface to destroy foreign substances, cell debris,
and prepared for the process of wound healing and
repair [36,37,43,44]. In respect of our results, the
finding is similar to those described by Singer and
Clark [43], so it seems macrophages were widely dis-
tributed in the body and played a role in the process
inflammation as the body’s reaction to foreign parti-
cles and bacteria. It has long been known that the mac-
rophages play an important role in inducing angio-
genesis in a manner secrete several factors: Tumor
necrosis factor-alpha, VEGF, angiogenin, urokinase,
and platelet-derived growth factor. The role of macro-
phages CD68 on angiogenesis (CD34 marker) in line
with research conducted by El-Rouby [44] showed
that the number of macrophages, which was widely
followed by an increasing amount of angiogenesis.
Conclusion
We concluded that the latex cream of J. curcas
possesses anti-inflammatory activity in wound heal-
ing process of mice skin.
Authors’ Contributions
MNS and AH designed and carried out the main
research work. The research was carried by DM,
UB, and CDI. The manuscript was written by MNS,
Darmawi, UB, CDI, and MH. All authors read and
approved the final manuscript.
Acknowledgments
Researchers would like to thank the Directorate
of Research and Community Service, the Directorate
General of Strengthening Research and Development,
Ministry of Research, Technology and Higher
Education Republic Indonesia that has provided
research funding Hibah Bersaing with contract num-
ber: 129/SP2H/LT/DRPM/III/2016.
Competing Interests
The authors declare that they have no competing
interests.
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