��

Vol. 45. No. 1 March 2012

Effect of Robusta coffee beans ointment on full thickness wound 
healing

Yorinta Putri Kenisa1, istiati2, and Wisnu Setyari J2
1Dental Student
2Department of Oral Biology 
Faculty of Dentistry, Airlangga University
Surabaya - Indonesia

abstract

Background: Traumatic lesions, whether chemical, physical, or thermal in nature, are among the most common lesion in the mouth. 
Wound healing is essential for the maintenance of normal structure, function, and survival of organisms. Experiments of Robusta coffee 
powder on rat-induced alloxan incision wound, clinically demonstrated similar healing rate with the povidone iodine 10%. No studies 
that look directly the effect of coffee extract in ointment form when viewed in terms of histopathology. Robusta coffee bean (Coffea 
canephora) consists of chlorogenic acid (CGA) and caffeic acid which are belived to act as antioxidant and take part in wound healing 
process. Purpose: The aim of this study was to identify the enhancement of healing process of full-thickness skin wound after Robusta 
coffee beans extract ointment application. Methods: Sample consisted of 20 Cavia cabaya treated with full-thickness with wounds and 
was given Robusta coffee beans extract ointment concentration range of 22.5%, 45%, and 90% except the control group which was 
given ointment base material. Animals were then harvested on the fourth day and made for histopathological preparations. Data were 
calculated and compared by one-way ANOVA test and LSD test. results: The study showed that Robusta coffee bean extract ointment 
can increase the number of lymphocytes, plasma cells, macrophages, fibroblasts, and blood vessels by the presence of chlorogenic 
acid (CGA) and Caffeic acid. Conclusion: In conclusion Robusta coffee bean extract ointment enhance the healing process of full-
thickness skin wound of Cavia cabaya.

Key words: Robusta coffee bean extract, the healing process, chlorogenic acid, caffeic acid

abstrak

latar belakang: Lesi traumatik, baik akibat rangsang kimia, fisik, atau termal, merupakan lesi yang paling umum terjadi di 
dalam rongga mulut. Penyembuhan luka yang terjadi ini penting untuk pemeliharaan struktur normal, fungsi, dan kelangsungan 
hidup organisme. Percobaan pemberian bubuk kopi Robusta terhadap luka sayatan pada tikus yang diinduksi aloksan, secara klinis 
menunjukkan tingkat penyembuhan yang sama dengan povidone iodine 10%. Namun belum ada penelitian yang melihat secara 
langsung pengaruh ekstrak kopi dalam bentuk salep bila dilihat dari segi histopatologi. Biji kopi Robusta (Coffea canephora) terdiri 
dari chlorogenic acid (CGA) dan caffeic acid yang dipercaya berperan sebagai antioksidan dan mengambil bagian dalam proses 
penyembuhan luka. tujuan: Tujuan penelitian ini adalah untuk mengidentifikasi peningkatan proses penyembuhan luka full-thickness 
pada kulit setelah pengaplikasian salep ekstrak biji kopi Robusta. Metode: Sampel terdiri dari 20 Cavia cabaya yang diberi perlakuan 
berupa luka sayat full-thickness pada kulit punggung dan diberi salep ekstrak biji kopi Robusta dengan beberapa konsentrasi, yaitu 
22,5%, 45%, dan 90%, sedangkan kelompok kontrol hanya diberi bahan dasar salep. Binatang coba kemudian dieksekusi pada hari 
keempat dan dibuat sediaan histopatologinya. Data dihitung dan dibandingkan dengan uji One-Way ANOVA dan uji LSD. hasil: Hasil 
penelitian menunjukkan bahwa salep ekstrak biji kopi Robusta dapat meningkatkan jumlah limfosit, sel plasma, makrofag, fibroblas, 
dan pembuluh darah yang dipengaruhi oleh chlorogenic acid (CGA) dan caffeic acid. Kesimpulan: Disimpulkan bahwa salep ekstrak 
biji kopi Robusta memiliki efek dapat meningkatkan proses penyembuhan luka full-thickness pada kulit Cavia cabaya.

Kata kunci: Salep ekstrak biji kopi Robusta, proses penyembuhan, chlorogenic acid, caffeic acid

Research Report



��Kenisa, et al.: Effect of Robusta coffee beans ointment

introduction

Wound can be defined as a disability or injury of living 
tissue caused by physical or thermal disturbance arising 
both pathologically and physiologically.1 Traumatic 
lesions, whether chemical, physical, or thermal in nature, 
are among the most common in the mouth. This lession to 
oral-sot tissue can occur due to accidental, iatrogenic, and 
factitious traumas. They may present as burns, ulcerations, 
and gingival recession.2 Bastone et al.,3 also described the 
aetiology of dental trauma from national and international 
studies as well as the different classifications currently 
used to report dental injuries. An English study determined 
the incidence of trauma to permanent incisors and related 
soft tissues as four cases/100 children/15 months, which 
was almost twice the incidence of Australian study. Based 
on those literatures, wound healing is essential for the 
maintenance of normal structure, function, and survival 
of organisms.4

Wound healing is a complicated pathophysiological 
process. Although mucosal wounds demonstrate accelerated 
healing compared to cutaneous wounds, both cutaneous and 
mucosal wound healing proceed through the same stages.5 
Wound healing consists of several stages, namely stage 
of acute inflammation, cell proliferation, and maturation. 
At the stage of proliferation, cell proliferative activity of 
fibroblasts in the lesion has a central role to begin the wound 
healing process. Increasing number of fibroblasts in the 
dermal showed the healing ability.6 Wound healing process 
may be hampered by the presence of reactive oxygen stress 
(ROS) produced by microbes or neutrophils in the wound 
area, through mechanisms that lead to DNA damage. This 
fact strengthens the opinion that the existence of local 
antioxidants in wound area became crucial factors that have 
promoted the acceleration of the healing process.7-9 

Several studies conducting the process of wound healing 
using natural materials have been widely applied. The use of 
natural materials done because it is easy to use, inexpensive, 
and has an adequate bactericidal or bacteriostatic effect.10 In 
addition, natural materials rarely cause adverse side effects 
compared with synthetic materials.11

One of these natural materials is Robusta coffee beans. 
Robusta coffee is widely spread on the island of Java, 
Sumatra, and Sulawesi. Price of this coffee is cheaper than 
other types of coffee and more resistant to diseases that 
attack the coffee plants. Robusta coffee contains various 
compounds including 42.3% sugars (polysaccharides), 
7.5% protein, 11% lipid, 2.4% caffeine, and 6.4% acids.12 
Also reported that the application powder of raw Robusta 
coffee on rat-induced alloxan incision wound, showed 
clinical cure rate similar to the application of povidone 
iodine 10%.13

The polyphenols of coffee, caffeic acid and chlorogenic 
acid (CGA), is believed to promote wound healing. Robusta 
coffee beans have higher number of these polyphenols 
than arabica coffee beans.14 Chlorogenic acid and caffeic 
acid have antioxidant properties that are significantly 
more potent than vitamin C and E.15 In addition to having 
antioxidant potential, Robusta coffee has also been 
investigated to have antibacterial ability against methicillin-
resistent staphylococcus aureus that can cause opportunistic 
infections on the injured area. Phenolic compounds in 
coffee also have been studied to reduce the effects of 
histamine, bradykinin, and leukotrienes as well as to reduce 
the activity of the complement system.16

Research on the potential ointment of Robusta coffee 
bean extract in dosage form in wound healing has not been 
reported. The extraction is done so that the active substances 
are needed can be taken optimally. The purpose of this 
study was to determine the potential of Robusta coffee bean 
extract ointment on the healing process of full-thickness 
wounds on the skin of male guinea pigs (Cavia cabaya) 
which was evaluated histopathologically.

materials and methods

This research is an experimental research laboratory. 
The material used is the ointment of Robusta coffee bean 
extract with a range of concentration of 22.5%, 45%, and 
90%. Robusta coffee beans that have been roasted and 
used as a powder, then extracted using ethanol solvent. The 
extract was mixed with an ointment base material (PEG 400 
and PEG 4000) and is based on the required concentration. 
This research used 20 male guinea pigs (Cavia cabaya), 
aged 2–3 months, and weighing 200–300 mg. Research 
subjects were divided into 4 groups each consisted of 5 
guinea pigs. Incision wound of 2.5 cm long with a depth of 2 
mm was created on the back skin of each guinea pigs using 
number 11 scalpel under the effect of 10% ether anesthesia 
by inhalation. Each treatment group was given ointment 
of Robusta coffee bean extract and a control group given 
only simple ointment base using a syringe at a quantity of 
2 cc. Then treated in a closed wound with sterile gauze 
and plaster bandages. All guinea pigs in each group were 
harvested on the fourth day using 10% ether as sedation. 
Back skin biopsies and subsequent histopathological 
preparations was done   using haematoxylin eosin (HE) 
staining. Then calculation of chronic inflammatory cells 
(macrophages, lymphocytes, plasma cells), capillary blood 
vessels, and fibroblasts were done.

The data obtained from histopathological examination 
is quantitative data obtained by calculating the number of 
cells and capillary blood vessels under light microscopy 

Correspondence: Wisnu Setyari J, c/o: Departemen Biologi Oral, Fakultas Kedokteran Gigi Universitas Airlangga. Jln. Mayjen Prof. 
Dr. Moestopo No. 47 Surabaya 60132, Indonesia. E-mail: wizn_zetya@yahoo.com



�� Dent. J. (Maj. Ked. Gigi), Vol. 45. No. 1 March 2012: 52–57

performed	on	five	different	fields	of	view	with	1000√	
magnification. These research data were analyzed with 
statistical tests of One-Way ANOVA and LSD.17

results

The largest number of lymphocytes present in the 
sample group which were given ointrment of 90% Robusta 
coffee beans extract, while the smalles number found in 
the control group. The largest amount of plasma cells 
present in the sample group which were given ointment of 
Robusta coffee beans extract concentration of 45%, while 
the smallest number found in the control group (Table 1).

The largest number of macrophages present in the 
control group, while the smallest number of groups 
present in concentrations of 22.5%. The largest number of 
fibroblasts present in the sample group which were given 
ointment of Robusta coffee bean extract concentration of 
45%, while the smallest number found in the control group. 
The largest number of capillaries present in the sample 
group which were given oinment of Robusta coffee bean 
extract concentration of 90%, while the smallest number 
found in the control group (Table 1).

Obtaining data on the number of cells and capillary 
blood vessels in each group performed One-Way ANOVA 
test. Before the One-Way ANOVA test, this study shows 
that the data are normally distributed after the Kolmogorov-

table 1. Distribution of mean and standard deviation of lymphocytes and plasma cells, macrophages, fibroblasts, and capillary blood 
vessels on the fourth day after treatment 

Groups X
 ± SD

Lymphocytes Plasma cells Macrophages Fibroblasts Capillary blood vessels
Control 13.60 ± 8.735* 2.60 ± 1.817 39.80 ± 21.394 148.20 ± 22.928* 206.00 ± 83.896

G1 (22.5%) 14.40 ± 3.435* 6.00 ± 4.899 19.20 ± 11,189 217.60 ± 57.051* 213.20 ± 68.766
G2 (45%) 25.60 ± 8.649* 8.80 ± 5.020 27.20 ± 8.927 271.00 ± 94.557* 219.80 ± 134.908
G3 (90%) 34.20 ± 19.136* 7.60 ± 2.702 27.80 ± 6.099 173.00 ± 22.226* 238.00 ± 63.075

Note: *: significant difference between groups

a b

c d

figure 1. Histopathological image on the group: A) control group, B) group 1 (22.5%), c) group 2 (45%), group 3 (90%).
 Note: 1) lymphocytes, 2) capillary blood vessels, 3) fibroblast, 4) macrophages, 5) plasma cells. (HE staining; magnification  

1000√;	Olympus	BX-50	microscope.	Pentax	optio	230;	Digital	Camera	2.0	megapixels).



��Kenisa, et al.: Effect of Robusta coffee beans ointment

Smirnov test statistic and homogeneous after Levene test. 
One-Way ANOVA test showed significant values   (p<0.05) 
in lymphocytes and fibroblasts. The average value of 
lymphocytes and fibroblasts in the treatment group differed 
significantly, whereas the other dependent variables such 
as plasma cells, macrophages, and capillaries found no 
significant difference.

table 2. LSD statistical test of significance figures on the 
number of lymphocytes and fibroblasts between 
groups

Concentrations 
of ointments 

Compared 
concentrations

Sig.
(Lymphocytes)

Sig.
(Fibroblasts)

Control 22,5% 0.697 0.074
45% 0.133 0.004*
90% 0.012* 0.505

22,5% Control 0.697 0.074
45% 0.065 0.161
90% 0.015* 0.238

45% Control 0.133 0.004*
22,5% 0.065 0.161
90% 0.273 0.016*

90% Control 0.012* 0.505
22,5% 0.015* 0.238
45% 0.273 0.016*

* the mean difference or significance value smaller than  
 0.05 (p <0.05)

To determine the effect of differences in test conducted 
further Post Hoc Test LSD. Significant differences 
in this table are expressed with an asterisk ‘*’ on the 
mean difference or significance value smaller than 0.05 
(p<0.05). In lymphocytes, the data showed significant mean 
differences in comparisons between the control group with 
group 3 and group 1 with group 3. Whereas in fibroblasts, a 
significant mean differences found in comparisons between 
the control group with group 2 and between group 2 with 
group 3.

discussion

Regeneration process can be seen from the cells that 
play a role during the wound healing process such as poli 
morpho nuclear (PMN) cells, lymphocytes, macrophages, 
plasma cells, fibroblasts, and capillary blood vessels. 
Observation of the results of this study was done the fourth 
day after treatment for acute inflammatory cells such as 
PMN, especially neutrophils which will soon be replaced 
by macrophages on the third day and granulation tissue 
which enter the slit incision. Gap is filled with granulation 
tissue and maximum vascularization on the fifth day. The 
observation of the results on this study was conducted on 
the fourth day so that all cells needed can be seen.4

This study used a range of 22.5%, 45%, and 90% 
concentration and is a preliminary study using ointment of 
Robusta coffee bean extract. Extraction of coffee was done 

so the active substances can be taken optimally. Ethanol 
was used as extraction solvent because it can withdraw the 
amount of phenolic acids higher than methanol and pure 
water.18 The ointment used are made of poly ethylen glycol 
(PEG) because PEG is chemically stable. Both PEG 400 and 
PEG 4000 used in this study are soluble in ethanol. PEG 
does not irritate skin and easy to clean by washing.19

Wound healing involves several mechanisms, such as 
inflammatory phase, proliferation, and maturation. In the 
inflammatory phase, the objectives are to stop the bleeding 
and clean the wound area of   foreign bodies, dead cells, and 
bacteria to prepare for the start of healing process. PMN cells 
migrate into the interstitial area to perform phagocytosis 
of foreign bodies and bacteria. However, wound healing 
is enhanced by the presence of stress ROS produced by 
PMN or microbial infection. If ROS are produced too 
much, it can cause cellular and DNA damage. CGA and 
caffeic acid are contained in Robusta coffee beans extract 
act as antioxidants to neutralize ROS which is the free 
radicals produced in the process of wound healing. ROS 
can increase lipid peroxidation which is a major cause of 
damage to the cell membrane so that it can damage the cell 
structure and function.20 Antioxidants have been reported 
to have a significant role in the process of wound healing 
and protect tissues from oxidative damage.21 Antioxidant 
mechanism is expected to protect cells that play a role in 
the process of wound healing. CGA and caffeic acid as 
antioxidants convert free radicals into stable products. The 
neutralized free radicals can not react on polyunsatured 
fatty acids (PUFAs) which generate alcoxyl and peroxyl 
radicals that responsible for the basic process of membrane 
cell lipid peroxidation.22 In the initial adhesion process, 
PMN adhere to the endothelium through the interaction of 
specific molecules such as selectin and glycosylated protein 
so that PMN ables to exit the endothelial transmigration as 
it is called an acute inflammatory process.23

This phase continues as chronic inflammatory cells into 
the injured area. Table 1 showed that the mean number of 
lymphocytes in group 3 is higher than the control group, 
group 1, and group 2. According to Hung et al.,24 CGA 
was shown to increase lymphocytes proliferation.24 It can 
be seen from the mean number of increased lymphocytes 
until the highest (90%). LSD test on lymphocytes showed 
that there were significant mean differences in comparisons 
between the control group with group 3 and group 1 with 
group 3 (Table 2). While results for the plasma cells in 
table 1 showed mean number of the highest plasma cells 
found in group 2 which did not differ significantly group 
3. Active substances contained in coffee, namely CGA, has 
been mentioned to have a role in increasing proliferation 
of lymphocytes. This is probably an indirect effect of 
plasma cells as these cells is the end product of activation 
of B lymphocytes activation that have differentiated, then 
plasma cell produce direct antibody against antigens in 
inflammation. 

Results of the next calculation is the amount of 
macrophage cells in control group which showed a higher 



�� Dent. J. (Maj. Ked. Gigi), Vol. 45. No. 1 March 2012: 52–57

mean than group 1, group 2, and group 3. This is presumably 
due to the treatment group, the phase of chronic inflammation 
will soon ends characterized by the declining number of 
macrophages and the beginning phase of proliferation. 
Increasing the mean number of macrophages seen in the 
treated group. Group 3 has the highest mean followed by 
group 2 and group 1. This is because CGA stimulates the 
mobilization of macrophages, may indirectly increase the 
ability of macrophage phagocytosis because it affects the 
secretion of IFN g that act as macrophage activators.22,24 
T lymphocytes which are activated by interaction with 
macrophages that present antigen fragments on the surface 
of cells can produce IFN g. These cytokines may activate 
macrophages so that macrophages release other cytokines 
to activate lymphocytes and causes inflammation where 
there is a focus of both these cells stimulate each other to 
destroy the antigen.

The next phase is the proliferative phase which 
involves the proliferation of fibroblasts, collagen 
synthesis, angiogenesis, granulation tissue formation, and 
epithelisation.25 An important first step in this phase is 
the improvement of microcirculation to supply oxygen 
and nutrients needed to fill the metabolic needs of tissue 
repair. Regeneration of new blood vessels (angiogenesis) 
is stimulated by hypoxic injury condition as well as several 
growth factors, particularly VEGF-A, FGF-2, TNF-b. At the 
same time, fibroblasts migrate into the wound in response 
to cytokines and growth factors produced by inflammatory 
cells, among which are macrophage.26 That activated 
macrophages can stimulate growth factors and cytokines 
(TGF-a, TGF-b, PDGF, VEGF -A, and IL-1) on the injured 
area. TGF-b plays a role in angiogenesis, reepithelisation, 
and connective tissue regeneration. TGF-b which are 
dominant in cutaneous wound healing is TGF-b1. TGF-b 
works by activating its receptor on the cell surface and 
transducing signal on target genes. Binding of a TGF-b to 
its type II receptor in concert with a type I receptor leads to 
formation of a receptor complex and phosphorilation of type 
I receptor. Thus activated, the type I receptor subsequently 
phosphorylates a receptor-regulated SMAD (R-Smad), 
allowing this protein to associate with Smad4 (Co-Smad) 
and move into nucleus. In the nucleus, the SMAD complex 
associate with a DNA-binding partner (Fast-1) and this 
complex binds to a specific enhancers in target genes so 
that it can activate the gene transcription.27,28 In the injured 
tissues, extracellular matrix molecules (ECM), namely 
tenascin-C, expressed during the process of tissue repair. 
Tenascin-C plays a role in proliferation and migration of 
fibroblasts. This molecule can induce phosphorylation of 
epidermal growth factor receptor (EGFR) and stimulates 
activation of mitogenic activated protein (MAP) kinase 
and mitogenesis of fibroblasts. In addition, tenascin-C can 
induce migration of fibroblasts through the activation of 
PLCg and m-calpain.29 Those growth factors and molecules 
play a role in cell proliferation and migration of fibroblasts 
so that the process of wound healing can be achieved. The 
counting results in graph 2 showed the mean number of 

fibroblasts in group 2 which is higher than the control group, 
group 1, and group 3. LSD test on fibroblast cells showed 
that there were significant mean differences in comparisons 
between the control group with group 2 and group 2 with 
group 3 (Table 2). The number of decreased fibroblasts 
in group 3 caused by the proliferation of fibroblasts cells 
which have reached the optimum effect at a concentration 
of 45%. The mean results of capillary blood vessels showed 
that the number of capillaries in group 3 is higher than the 
control group, group 1 and group 2. An increasing number 
of these occurred with increasing concentrations of the 
ointment. This occurs indirectly as the influence of several 
growth factors such as VEGF-A, FGF-2, TNF-b that are 
produced both by macrophages and fibroblasts. TGF-b1 
produced by macrophages can also induce up-regulation 
of growth factor for angiogenesis such as VEGF.27

Fibroblasts are actively moving from the network 
around the wound into the wound area, proliferate and 
issue some substances (collagen, elastin, hyaluronic acid, 
fibronectin, and proteoglycans) that play a role in forming 
new tissue. Collagen is a protein substance that increase the 
surface tension of the wound.25Other phenolic compounds 
in coffee, namely caffeic acid, has also been studied to play 
a role in the healing process by stimulating the synthesis of 
collage-like polymer by fibroblasts.6 Increased amount of 
collagen that add strength to the wound surface can avoid 
the possibility of opened wound.26

In the results of data analysis, the highest levels of 
Robusta coffee bean extract ointment (90%) showed 
the highest value on the mean number of lymphocytes, 
macrophages, and capillary blood vessels, but not in 
plasma cells and fibroblasts, although it is higher than the 
concentration of 22.5%. While coffee bean extract 45% 
concentration ointment showed the highest value on the 
average number of plasma cells and fibroblasts compared 
to ointment of coffee bean extract 90%. Overall, Robusta 
coffee bean extract 45% ointment can give a good effect 
on wound healing process because at this concentration 
the number of fibroblasts increased significantly compared 
with the control group. It can be concluded that ointment 
of Robusta coffee been extract could enhance skin wound 
healing process of Cavia cabaya.

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