Keywords
open wound - healing - histological assay - Moringa oleifera Lamarck
Introduction
Palatal keratinized gingiva is a common site to obtain a free soft-tissue graft for
gingival augmentation procedures, but often leads to postoperative morbidity because
of the open wound.[1] Inconvenience caused by pain, postsurgical bleeding, and necrotizing tissue may
arise after harvesting the palatal graft, therefore, it is necessary to enhance healing
time when the palatal donor site is required for repeated procedures.[2]
After harvesting a palatal free graft, an open wound devoid of epithelium is created,
extending deep into the connective tissue and/or bone. This wound type requires a
significant amount of new tissue formation with secondary intention healing.[3]
[4] Postsurgical mechanical plaque control may be difficult to perform, thus compromising
the healing process.[5]
[6] A variety of antimicrobial agents have been used to enhance epithelial healing process,
thereby preventing infection and chronicity of the wound. Unfortunately, microbes
change their metabolism and genetic structure to acquire resistance against the antimicrobial
agents used in the treatment of infections.[7]
[8] To overcome microbial drug resistance, many studies were performed to develop alternative
and novel drugs.
In Indonesia, plants has one of the most extensive floras in the world with more than
20,000 species and a large proportion of the population utilizes medicinal plants
as remedy of infectious diseases.[9] Antibacterial, anti-inflammatory, and wound healing activity of plant extracts of
Indonesian medicinal plants have been reported in the literature, but the vast majority
has yet to be investigated.
One of the most studied plant is Moringa oleifera Lamarck., native to the Asian subcontinent and Arabia, often considered as an indigenous Indonesian
species.[10] Known as Kelor by local people, it has been used by Indonesians in Sulawesi, Java and East Nusa
Tenggara as fertilizers, fodder, food, fruit ripening, glue, medicinal, rituals, seed,
skin care, water purification, and support for vine crops.[11] The leaves of this plant have been reported to contain, vitamin C, β-carotene, protein,
calcium, potassium, as well as flavonoids, phenolics and carotenoids and have antimicrobial,
antifungal, and wound healing activities.[12]
[13]
M. oleifera extract has been documented to have antibacterial effect against oral pathogens,
anti-biofilm, and antifungi.[14]
[15]
[16]
[17] Furthermore, treatment by M. oleifera extract can reduce IL1-β and TNF-α increased in the gingival tissue in a model of
periodontitis in rat.[18]
This study was designed to explore the healing effects of topically applied gel prepared
from M. oleifera Lamarck leaves extracts in rat palatal wound.
Materials and Methods
The experimental design and protocols were reviewed and approved by Ethics Committee
in Hasan Sadikin Hospital, Bandung, West Java-Indonesia (33/UN6C10/2018).
Plant Material and Extract Preparation
M. oleifera Lamarck (Moringaceae) leaves were collected in April 2017 from Kefamenanu, East Nusa
Tenggara at 9°26′48″S 124°28′41″E and at 50 m above sea level. It was authenticated
with collection number 049/HB/02/2017 deposited in Herbarium of Plant Taxonomy Laboratory,
Department of Biology, Faculty of Math and Nature Sciences, Universitas Padjadjaran
Jatinangor, West Java, Indonesia.
Preparation of Ethanolic Extracts of M. oleifera
The fresh leaves were collected, surface sterilized, sun dried for 7 days on ground.
The powdered material (1.5 kg) was exhaustively extracted (3 cycles/h) with 95% ethanol
in Soxhlet apparatus by continuous hot extraction. After each extraction, the solvent
was recovered using flash evaporator, and the extract was concentrated under reduced
pressure. Then the crude extract was dissolved in the solvent and stored in air-tight
glass bottles at 4°C and later redissolved in their respective solvents to the desired
concentrations for the various experiments.
Gel Preparation
M. oleifera leaf extract gel 2 and 4% were made by dissolving 1 gram and 2 grams extract in 5
mL of aquades with an ultrasonic instrument and subsequently mixed with 50 grams gel
basis (HPMC 4%) as vehicle until it became homogeneous. Ethanol extract and gel were
prepared in the Laboratory of Pharmacy, Universitas Jenderal Ahmad Yani, Cimahi, West
Java, Indonesia.
Animals
Sixty male Sprague–Dawley rats (200–300 g) were used to carry out the study. One week
before the experimental procedures, the animals were housed and adapted at air-conditioned
animal laboratory room (22 ± 3°C) with 12-hour light and dark cycle. The rats were
fed with commercial normal rodent pellet and filtered water ad libitum. All of the
animals received humane care according to the criteria outlined in the Guide for the
Care and the Use of Laboratory Animals prepared by the National Academy of Science
and published by the National Institute of Health. The ethics regulations were followed
in accordance with national and institutional guidelines for the protection of the
animals’ welfare during experiments.[19] All of the experimental procedures were performed in Laboratory of Experimental
Animal, Faculty of Medicine, Universitas Jenderal Ahmad Yani, Cimahi, West Java, Indonesia.
Wound Induction and Experimental Animal Groups
The animals were randomly divided into four experimental groups (n = 15 per group) as follows:
-
Group I: wounding and Moringa leaf extract gel 2%,
-
Group II: wounding and Moringa leaf extract gel 4%,
-
Group III: wounding and povidone iodine gel 10%, and
-
Group IV: wounding and HPMC 4% (vehicle only).
The animals were anesthetized with an intraperitoneal injection of 10% Ketamine and
a 4-mm mucosal wound was made on a central area of hard palate to the depth of the
periosteum using a disposable round stainless steel blade designed for punch biopsy
(Mentok Co., Ltd., India) exposing a circular area of bare bone and gingiva was separated
by periosteal elevator.[20] Each group received allocated regimen immediately after the wounding procedures
by researcher's assistant once daily for 14 days. The animals were sacrificed with
decapitation, assessments were performed at days 0, 3, 7, 10, and 14 days after wound
induction.
Histological Assay
Tissue specimen were excised and immersed at days 0, 3, 7, 10, and 14 after wound
induction and preserved in 10% neutral buffered formalin before tissue processing
procedures. The tissue sections embedded in paraffin wax were sectioned in 5 μm thickness,
followed by dewaxed and rehydrated conventionally. These sections were then stained
with hematoxylin and eosin (H&E) for fibroblast assessment, while Masson's trichrome
staining was employed to reveal collagen deposition. The stained samples viewed under
the light microscope (Olympus BX 41, United States) at ×100 magnification and then
processed with ImageJ software (National Institute of Health, United States).
Statistical Analysis
All data were subjected to statistical analysis using SPSS 20.0 (SPSS, IBM, New York,
NY, United States). Shapiro–Wilk test was employed to determine normality of the data.
All values were represented as means ± SD and were analyzed using analysis of variance
(ANOVA) then by Duncan's post-hoc for multiple comparisons (p < 0.05 was considered significant).
Results
All 60 rats survived the surgical procedures with no complications. Findings on each
group were evaluated and histological differences were compared between control and
experimental groups of section.
To test the normality of the data, Shapiro-Wilk test was employed, all data was normally
distributed. One way analysis of variance (ANOVA) was then performed ([Tables 1]
[2]), followed by Duncan's post-hoc to measure specific differences between pairs of
means ([Tables 3]
[4]).
Table 1
Mean fibroblast population after wound
|
HPMC
|
PI
|
Moringa (2%)
|
Moringa (4%)
|
p-Value
|
|
Abbreviations: HPMC, hydroxypropyl methylcellulose; PI, povidone iodine; SD, standard
deviation.
Note: ANOVA test.
|
|
Day 3 (±SD)
|
20 (2)
|
18 (3)
|
24 (3)
|
32 (5)
|
≤ 0.00a
|
|
Day 7 (±SD)
|
26 (5)
|
39 (3)
|
36 (5)
|
39 (5)
|
≤ 0.00a
|
|
Day 10 (±SD)
|
30 (5)
|
41 (5)
|
36 (4)
|
70 (5)
|
≤ 0.00a
|
|
Day 10 (±SD)
|
23 (5)
|
35 (5)
|
32 (4)
|
51 (6)
|
≤ 0.00a
|
Table 2
Collagen deposition (%) after wound
|
HPMC
|
PI
|
Moringa (2%)
|
Moringa (4%)
|
p-Value
|
|
Abbreviations: HPMC, hydroxypropyl methylcellulose; PI, povidone iodine; SD, standard
deviation.
Note: ANOVA test.
aStatistically significant
|
|
Day 3 (±SD)
|
37.09 (4.45)
|
28.69 (4.44)
|
43.51 (4.93)
|
45.45 (2.28)
|
≤ 0.00a
|
|
Day 7 (±SD)
|
40.90 (5.11)
|
48.01 (3.87)
|
45.94 (5.64)
|
63.07 (5.27)
|
≤ 0.00a
|
|
Day 10 (±SD)
|
66.67 (4.10)
|
68.69 (4.18)
|
69.50 (4.79)
|
68.61 (2.85)
|
= 0.276
|
|
Day 14 (±SD)
|
68.78 (3.97)
|
71.56 (3.91)
|
74.80 (6.25)
|
74.02 (5.68)
|
= 0.08a
|
Table 3
Duncan post-hoc analysis for fibroblast density
|
Abbreviation: HPMC, hydroxypropyl methylcellulose.
|
|
Subset for α= 0.05
|
|
Day 3
|
1
|
2
|
|
Povidone iodine
|
18.27
|
|
|
HPMC
|
20.07
|
|
|
Moringa (2%)
|
|
24.80
|
|
Moringa (4%)
|
|
|
|
Significant
|
0.152
|
1.000
|
|
Day 7
|
1
|
2
|
|
HPMC
|
25.67
|
|
|
Moringa (2%)
|
|
35.73
|
|
Moringa (4%)
|
|
38.93
|
|
Povidone iodine
|
|
|
|
Significant
|
1.000
|
0.067
|
|
Day 10
|
1
|
2
|
3
|
4
|
|
HPMC
|
30.47
|
|
|
|
|
Moringa (2%)
|
|
36.40
|
|
|
|
Povidone iodine
|
|
|
41.00
|
|
|
Moringa (4%)
|
|
|
|
70.27
|
|
Significant
|
1.000
|
1.000
|
1.000
|
1.000
|
|
Day 14
|
1
|
2
|
|
|
|
HPMC
|
23.07
|
|
|
|
|
Moringa (2%)
|
|
32.13
|
|
|
|
Povidone iodine
|
|
34.67
|
|
|
|
Moringa (4%)
|
|
|
|
|
|
Significant
|
1.000
|
0.194
|
|
|
Table 4
Duncan post-hoc analysis for collagen density
|
Subset for α= 0.05
|
|
Day 3
|
1
|
2
|
3
|
|
Abbreviation: HPMC, hydroxypropyl methyl.
|
|
Povidone iodine
|
28.6907
|
|
|
|
HPMC
|
|
37.0958
|
|
|
Moringa (2%)
|
|
|
43.5116
|
|
Moringa (4%)
|
|
|
45.4575
|
|
Significant
|
1.000
|
1.000
|
0.206
|
|
Day 7
|
1
|
2
|
3
|
|
HPMC
|
40.9063
|
|
|
|
Moringa (2%)
|
|
45.9404
|
|
|
Moringa (4%)
|
|
48.0197
|
|
|
Povidone iodine
|
|
|
63.0797
|
|
Significant
|
1.000
|
0.262
|
1.000
|
|
Day 14
|
1
|
2
|
|
|
HPMC
|
68.7897
|
|
|
|
Povidone iodine
|
71.5617
|
71.5617
|
|
|
Moringa (4%)
|
|
74.0247
|
|
|
Moringa (2%)
|
|
74.8063
|
|
|
Significant
|
0.139
|
0.102
|
|
In the presence of Moringa 4%, the growth of fibroblasts and collagen deposition were
found to be significantly higher (p < 0.05) than in the other group tested.
Three days after injury, the Moringa–treated wound's fibroblasts significantly repopulated the wound area compared with
povidone iodine or HPMC groups.
Seven days after injury, wounds treated with Moringa and povidone iodine displayed significantly improved fibroblasts proliferation compared
with HPMC group ([Table 1]).
Ten days after injury, fibroblasts were found to be significantly higher in Moringa 4%-treated groups (p < 0.05) than in the other groups tested ([Fig. 1]).
Fig. 1 Fibroblast population after wound. HPMC, hydroxypropyl methylcellulose; PI, povidone
iodine.
Histological section showed that in Moringa-treated wounds, fibroblasts migrated faster toward the open wounds than povidone
iodine or HPMC-treated ([Fig. 2]).
Fig. 2 Fibroblast at day 10 (magnification 100X). HPMC, hydroxypropyl methylcellulose.
An increased formation of new collagen into the wound area was observed based on the
percentage of brightness from Image-J program for all groups tested from days 3 to
14. This behavior was best observed in the Moringa 2 and 4% groups ([Figs. 3])
Fig. 3 Collagen density after wound. HPMC, hydroxypropyl methylcellulose; PI, povidone iodine.
Fig. 4 Collagen deposition at day 7 (Magnification 100X). HPMC, hydroxypropyl methylcellulose.
Discussion
The present study was conducted to test the effect of topical application of M. oleifera leaves extract on the healing process of excisional wounds with connective tissue
deficiency that heals by secondary intention. In wound healing, good tissue growth
was defined as a tissue rich in fibroblasts and dense in newly synthesized collagen
determined by Masson's trichrome.[21]
The findings of this present study revealed that the rate of fibroblast proliferation
and collagen deposition of the wound were significantly superior following the application
of both 2 and 4% Moringa leaves extract gel, and slower when povidone iodine gel and vehicle only was applied.
M. oleifera extract has been shown to have accelerating effect on wound healing in skin. The
ethanol and ethyl acetate extracts of seeds showed significant antipyretic activity
in rats, whereas ethyl acetate extract of dried leaves showed significant wound healing
activity (10% extracts in the form of ointment) on excision, incision, and dead space
(granuloma) wound models in rats.[22] The aqueous extract of M. oleifera promotes wound healing significantly and wound healing-suppression action of dexamethasone
could be overcome.[23] Activation of fibroblasts, endothelial cells, and macrophages are keys of wound
healing in which body cells response to injury. The restoration of structure and function
in the wound site is determined by fibroblast proliferation.[24] Therefore, therapeutic bioactive agents that are able to stimulate fibroblast growth
and proliferation may be able to improve or promote wound healing as in the case of
the present study, ethanol extract of M. oleifera leaves prepared in gel was demonstrated to enhance the proliferation of fibroblast
and collagen deposition in palatal rats.
Unlike skin surface, wound located in the oral cavity is surrounding by the unique
environmental challenge for the epithelial healing of oral wounds produced during
various periodontal procedures, since the oral environment cannot be sterilized from
oral bacteria or plaque formation. Therefore, wound surface must be protected from
the external environment or infection after periodontal surgery. The donor sites require
2 to 4 weeks to heal with secondary intention resulting in experience of pain and
postoperative bleeding. In the present study, more fibroblast and collagen deposition
were seen at palatal donor site at days 10 and 14 in which Moringa gel was given.
The mechanisms through which the M. oleifera leaves extract accelerates wound healing were further explored by examining whether
this extract increased fibroblast migration and collagen deposition in wound tissue.
Collagen fibers reached the highest deposition in wounds treated with M. oleifera leaves extract 4%. The dispersed blue stain indicated thicker and more mature tissue
collagen formation in wounds treated with this extract, suggesting that exposure of
M. oleifera leaves extract maintained connective tissue architecture through fibroblast migration
and ultimately collagen deposition.
Plant used as phytochemical may inhibit bacterial growth by different mechanisms than
the presently used antibiotics. Peptides content in M. oleifera has an action on membrane disruption of several species of Staphylococcus including MRSA, as well as Streptococcus sp, Eschericia coli and Enterococcus faecalis.[25] Elgamily et al showed antimicrobial and antifungal potential effects of Moringa leaves extract against oral pathogens.[17] The leaves of M. oleifera plant are known to contain phytochemical compounds as flavonoids, saponins, tannins,
and other phenolic compounds that have antimicrobial activities.[26]
[27]
[28] This would explain sites given Moringa extract were protected from microbial or fungal challenge from oral cavity that may
compromise the wound healing.
This study showed wound healing effect of M. oleifera leaves extract was comparable with those of povidone iodine in positive control group.
Bioactive fraction of M. oleifera containing Vicenin-2 compound may contribute to enhance faster wound healing in vitro
as shown by Muhammad et al (2013).[29]
In conclusion, within the limitations of the present study: (1) it is confirmed that
M. oleifera leaves ethanol extract stimulates fibroblast and collagen deposition during the initial
phase of wound healing in palatal rats, (2) the leaves extracts can be used to formulate
new dental products to accelerate wound healing in oral cavity owing to their antibacterial
and anti-inflammatory potentials. However, further studies are required to clarify
the optimal concentration and physical stability before its clinical application.
