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Therapeutic Applications of Garlic and Turmeric for the Diabetic Wound

Healing in Mice

Article in Journal of burn care & research: official publication of the American Burn Association · November 2022
DOI: 10.1093/jbcr/irac169

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 ORIGINAL ARTICLE

Therapeutic Applications of Garlic and Turmeric for the

Diabetic Wound Healing in Mice

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Muhammad A. Farooq, MPhil* Shaukat Ali, PhD*, Rida Sulayman, MPhil* Ali Hassan, MPhil*
Hafiz M. Tahir, PhD* Hafsa Shahzad, MPhil* Umaima Fiaz, MPhil* Tafail A. Mughal, PhD†
Irfana Liaqat, PhD* Samaira Mumtaz, MPhil* Tooba Nauroze, MPhil* and Shumaila Mumtaz, PhD*

Diabetes is involved in delayed wound healing that can be cured by natural products such as garlic, turmeric, and
fibroin extracts. Alloxan monohydrate is used for inducing diabetes in mice. The percent wound contraction of
garlic (150 mg/ml), turmeric (100 mg/ml), and fibroin (50 mg/ml), individually and in combinations garlic
(150 mg/ml) + fibroin (50 mg/ml), turmeric (100 mg/ml) + fibroin (50 mg/ml), garlic (150 mg/ml) + turmeric
(100 mg/ml), and garlic (150 mg/ml) + turmeric (100 mg/ml) + fibroin (50 mg/ml) was checked by evaluating
the healing time, % wound contraction and histological analysis. The serum level of MMPs (MMP 2, MMP7,
MMP 9), pro-inflammatory cytokines (TNF-α, IL-6, IL-8), and TIMPs were evaluated. With the combination of
three extracts (Ga+Tu+Fi) garlic (150 mg/ml), turmeric (100 mg/ml) and fibroin (50 mg/ml), wounds healed
in 12 days and had 97.3 ± 2.2% wound contraction. While the positive control (polyfax) and diabetic control
(saline) wounds healed in 17- and 19-days with wound contraction of 96.7 ± 1.4% and 96.3 ± 1.1%, respectively.
Histological analysis showed that the combination of Ga+Tu+Fi exhibited an increase in the growth of collagen
fibers, fibroblasts number, and keratinocytes, and lessened inflammation of blood vessels. The combination
of Ga+Tu+Fi significantly alleviated the serum concentration of TNF-α (14.2 ± 0.7 pg/ml), IL-6 (10.0 ± 1.0
pg/ml), IL-8 (16.0 ± 1.5 pg/ml), MMP2 (228.0 ± 18.1 pg/ml), MMP7 (271.0 ± 9.9 pg/ml), and MMP9
(141.0 ± 5.3 pg/ml) to diabetic control. The level of TIMPs (193.0 ± 9.1 pg/ml) was increased significantly with
respect to diabetic control. We conclude that the combination of these biomaterials possessed high regenerative
and healing capabilities and can be an effective remedy in the healing of chronic wounds in diabetic patients.

Diabetes also severely affects the wound healing process by
disturbing the several physiological processes of the body in-
volved in this process.1 Diabetes influences the normal wound
healing process in various ways. The normal wound healing
process possesses the following steps such as inflammation,
cell proliferation, re-epithelization, neovascularization, and
tissue remodeling.2 In the diabetic wound, prolonged inflam-
mation, disturbed circulation, and a low supply of nutrients
delay the normal wound healing process.3 Hyperglycemia
enhances oxidative stress and chronic inflammation due to
the presence of pro-inflammatory cytokines, which affects
wound healing.4
During diabetes wound healing, the transition between M1
and M2 does not occur. M1 macrophage levels remain high
due to this reason pro-inflammatory factors present in high
amounts and lead to impaired and delayed wound healing.
The pro-inflammatory factors also increase the protease
matrix metalloprotease-9 (MMP-9) which also promotes
From the *Applied Entomology and Medical Toxicology Laboratory, Department
of Zoology, Government College University, Lahore, Pakistan; †Department of
Zoology, Women University of Azad Jammu Kashmir, Bagh, Pakistan
Conflicts of Interest and Source of Funding: The authors have no competing
interest and did not receive any funding for this study.
Address correspondence to Dr. Shaukat Ali, PhD, Department of Zoology,
Government College University, Lahore, Pakistan. Email: dr.shaukatali@gcu.
edu.pk
© The Author(s) 2022. Published by Oxford University Press on behalf of the
American Burn Association. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com.
https://doi.org/10.1093/jbcr/irac169
impaired wound healing with diabetes.5 The high level of
M1 macrophages in diabetic patients abates apoptosis. As a
result, clearance of neutrophils by apoptosis is halted which
is necessary for the transition of M1 macrophages into M2
macrophages.6
In diabetes, the angiogenesis process is disturbed due to
various complications. The high level of glucose in the blood
of diabetic patients causes various micro and macrovascular
complications that ultimately influence normal angiogenesis.7
In the diabetic wound, the deficits in macrophages reduce the
ability of angiogenesis. The level of VEGF-A and mRNA were
decreased in wounds of db/db mice as compared to healthy
mice. VEGF-A enhanced the process of wound closure of db/
db mice when they were treated with it.8
During the remodeling phase, balanced MMPs and their
inhibitors TIMPs level are important because it plays a key
role in the reorganization of ECM. But in diabetes due to
hyperglycemia, the balance between MMPs and TIMPs is
disturbed which leads to impaired modification of ECM and
chronic wound formation.9
Delayed and impaired diabetic wound healing is a serious
health challenge globally nowadays. Various medical plants
and bioproducts are used for the healing of diabetic as well
as non-diabetic wounds. These natural products and plants
show positive results for the wound healing process along
with fewer side effects. Because these products are less toxic as
compared to allopathic medicines.10
Various physiological disorders like diabetes and wound
healing are treated by using garlic.11 For healing diabetic

1

2  Farooq et al
wounds, aged garlic solutions (AGS) were used in chicken
wound healing. AGS accelerated the healing process by
re-epithelialization and angiogenesis.12 Allicin, a component
of garlic, enhances the wound process by activating fibroblast
cells.13
The rhizome of turmeric has radical scavenger, anti-in-
flammatory, antioxidant, and anti-infectious activities, these
activities are important for the wound healing process.14–16
Curcumin also enhances the synthesis of the growth factors
that are involved in the wound healing process.17 Curcumin
also promotes cutaneous wound healing because it involves
tissue formation, tissue remodeling, collagen deposition,
and granulation.18 Vascular density, epithelial regeneration,
and fibroblast proliferation in the wound are promoted
by using curcumin.19 Other components of turmeric like
turmerones, elemene, furanodiene, Curdione, bisacurone,
and germacrone, also have anti-inflammatory,20,21 antioxi-
dant,22,23 and antimicrobial24–26 activities. That’s why turmeric
is used for the treatment of diabetic wound healing.27
Silk fibroin is used in various drugs that are used for wound
healing. Silk fibroin promotes the proliferation, cell growth,
and migration of cell types during various phases of the wound
healing process.28 Fibroin also promotes the various other
processes involved in wound healing like cellular adhesion, and
re-epithelialization, wound contraction, collagen formation,
and angiogenesis.29 During the inflammation phase of the
wound, silk fibroin suppresses the pro-inflammatory cytokines,
as a result, it protects the cells and tissues during the healing.
Along with all these activities during the healing process, silk
fibroin also deactivates the apoptotic pathways of the cells.28
Due to all these properties of silk fibroin, various scientists
used silk fibroin and its derivative for diabetic wound healing
and it showed good, cheaper, safer, and effective results.30–32
As discussed earlier, bioproducts, garlic (Allium sativum),
turmeric (Curcuma longa), and fibroin, use to heal diabetic
wounds. These natural products have fewer side effects and
are easily approachable. That’s why, in current studies, garlic,
turmeric, fibroin, and their combinations were used to ob-
serve the wound healing in diabetic mice by determining the
percent wound contraction, histological analysis, and bio-
chemical parameters.
METHODS
Ethical Statement
All the experiments were conducted following approval
from the Institutional Bioethics Committee (Ref. # GCU/
IIB/869) on 10th January 2022, GC University Lahore,
Pakistan.
Mice Handling and Rearing
In the current study, fifty Swiss female albino mice (8 weeks
old) were used as the experimental model. They were reared
in standard plastic cages in the Animal Housing facility of
the Zoology Department, Government College University,
Lahore. They were kept at a suitable temperature (22°C),
proper humidity (45–46%), and ventilation conditions. Ad
libitum conditions were given (adequate food and water
supply). They were provided with 12 hour light-dark cycle.
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Albino mice weighing around 25–30 g, at the age of 8 weeks,
were habituated for about one week before the experimental
manipulation.
Diabetes Induction in Mice
After acclimatization, a single dose of alloxan monohydrate
was intraperitoneally injected into the mice to induce diabetes
type 1. The dose was prepared by alloxan monohydrate in
brine with 150 mg/kg dosage given as per body weight.33 10%
Glucose solution was provided to the mice for drinking after
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the injection of alloxan monohydrate. An electric glucometer
is used to measure the glucose level in blood within the gap
of one week, it is done by pricking the tail of mice with an
On-call extra meter and strips. Those mice selected for fur-
ther studies had blood glucose levels of ≥150 mg/dl and
≤250 mg/dl. Before the initiation of the experiment, albino
mice with high levels of blood glucose were not included in
further studies.34
Formation of Excision Wound in the Skin of Mice
After the induction of diabetes, mice were divided into ten
groups each group comprising 5 mice. Anesthesia was pre-
pared by adding xylazine (1.6 mg/ml) and ketamine (16 mg/
ml) in saline solution that was given intraperitoneally before
the formation of the wound. Each mouse received 10 µl anes-
thesia per gram of its body weight (10 µl anesthesia contained
16 µg xylazine and 160 µg ketamine). The fur on the dorsal
surface of mice was shaved completely via an electrical shaver.
Two full thickness excision wounds were formed on the shaved
surface of the mouse via a biopsy punch device. The diameter
of each wound was 6 mm. All the surgical interventions were
carried out under sterile conditions. All mice received their re-
spective treatments, at least once a day, a day after the wound
had formed till complete healing. Irritations, the color of skin,
and body weight were observed and recorded on daily basis.35
Extraction of Fibroin
Degumming. Bombyx mori (silkworms) produced silk
cocoons, these cocoons were used to obtain silk fibers. For the
degumming of raw silk fibers, they were heated at 100°C in
0.5% NaHCO3 solution for 1 hour, then rinsed with distilled
water three times and dried in the oven overnight.
Dissolution of Silk Fibers. 100 mg of degummed fibers were
dissolved in a solvent, comprising 1% calcium chloride: eth-
anol: distilled water (1:2:8), with constant stirring for 6–8
hours at 80°C. 8 mM urea was dissolved in CaCl2 solvent to
obtain the complete dissolution of silk fibers.
Dialysis. For the removal of extra chemicals from dissolution,
a cellulose dialysis membrane was used to perform the dialysis
for three days in distilled water. Then silk fibroin solution was
sonicated at 20 kHz: 400W for 1hr and then obtained the silk
fibroin powder by lyophilization.34
Extraction of Garlic
For the extraction of garlic (Allium sativum), garlic powder
(300 mg/ml) was dissolved in ethanol. Shaked occasionally
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Volume XX, Number XX
for 48 hours. Then rotary hours. Then extract was filtered by
using cotton wool. Then the small quantities of benzene were
added to wool and heated to purify and again filtered by using
wool gauze. Then stored the extract at 4°C in an airtight con-
tainer for further use.36
Extraction of Turmeric
The rhizome of turmeric (Curcuma longa) is cut into pieces
and ground into the dried powder. To obtain the desirable
powder passed it through sieve number 60 (pore size 250 µm).
The powder was kept in an airtight jar. Then dissolved the
powder into the ethanol with a ratio of 1:10 (100 mg/ml) and
placed it in the shaker for 7 days. It was filtered and distilled
under a vacuum to get concentrated ethanolic extract. Then
lyophilized it to get powder form and stored it in the desic-
cator for further use.37
Preparation of Extracts Solutions and Their
Combinations
Prepared the garlic extract solution (150 mg/ml), turmeric
extract solution (100 mg/ml), and fibroin solution (50 mg/
ml). Four combinations were prepared by using an equal
volume of each extract. These combinations are the following:
Ga+Fi = 150 mg/ml garlic extract and 50 mg/ml fibroin.
Tu+Fi = 100 mg/ml turmeric and 50 mg/ml fibroin.
Ga+Tu = 150 mg/ml garlic extract and 100 mg/ml
turmeric.
Ga+Tu+Fi = 150 mg/ml garlic extract, 100 mg/ml tur-
meric, and 50 mg/ml fibroin.
Figure 1. Schematic diagram of experimental design.
Farooq et al  3
Experimental Design
Fifty Swiss albino female mice (25–30 g body weight) were
categorized into two major groups, Normal that is, non-
diabetic control (N = 5, named “A”) and diabetic group
(N = 45, named “B”), induced diabetes with alloxan. In all
the groups of mice, superficial wounds were introduced via
biopsy punch on the dorsal side of the mice after shaving
the hair. After this treatment, the diabetic group (B) was di-
vided further into nine subgroups, each containing 5 mice.
Group A, Normal, Non- diabetic control (named as “N”),
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and diabetic control B1 (named “DC”) groups were treated
with saline. Positive control B2 (named “PC”) was given
an ointment (polyfax). 150 mg/ml garlic extract solution
(150 µg/µl of each dose) was applied to the wound once a
day till complete healing. Group B3 was named “Ga”. Group
B4 and B5 have received 100 mg/ml turmeric (100 µg/µl
of each dose) extract solution (named “Tu”) and 50 mg/
ml fibroin (50 µg/µl of each dose) solution (named “Fi”)
extracts, respectively. Applied the combination of 150 mg/
ml garlic extract (75 µg/µl of each dose) and 50 mg/ml fi-
broin (25 µg/µl of each dose) (named as “Ga+Fi”) to the B6
group. Group B7 was given 100 mg/ml turmeric (50 µg/µl
of each dose) extract along with 50 mg/ml Fibroin (25 µg/µl
of each dose) (named as “Tu+Fi”). Group B8 was treated with
a combination of 150 mg/ml garlic (75 µg/µl of each dose)
and 100 mg/ml turmeric (50 µg/µl of each dose) extract
(named as “Ga+Tu”). Group B9 was given 150 mg/ml garlic
(50 µg/µl of each dose), 100 mg/ml turmeric (33 µg/µl of
each dose), and 50 mg/ml fibroin (16 µg/µl of each dose) ex-
tract in combination (named as “Ga+Tu+Fi”) as Figure 1. All

4  Farooq et al
these formulations were applied (100 µl/mice) superficially
on their wounds once a day till complete healing.
Percent Wound Contraction
After the formation of the wound, the diameter of each wound
was measured with a gap of one day until wounds were com-
pletely healed. The contraction represented percent wound
healing and epithelialization time was observed after complete
healing. The rate of healing or percentage contraction was
measured by using the following formula:38
Initial wound area − Wound area on a specif ic day × 100
= .
Initial wound area
Histological Analysis
Skin tissue of one mouse from each group was removed at
post-wounding day 10. Then used the 10% buffered formalin
(pH = 7) to preserve these tissues, and hematoxylin-eosin, and
Masson’s trichrome staining method were used.39 In the end,
observed the prepared slides of the tissues under the com-
pound microscope using low magnification power for the
histological analysis. In the histological slides, neutrophils,
keratinocytes, fibroblast cells, collagen fibers, and blood
vessels were observed at 10×.
Analysis of Biochemical Parameters
The blood of mice was collected via cardiac puncture when
the wounds were completely healed in each group. Then,
biomarkers in the blood serum that is, pro-inflammatory
cytokines (TNF-α, IL-6, and IL-8) matrix metalloproteinases
(MMP 2, MMP7, and MMP 9), and tissue inhibitor matrix
metalloproteinases (TIMPs), were analyzed by using enzyme-
linked immunosorbent assay (ELISA) Kits.40
Statistical Evaluations
For statistical analysis, the normality of the data was assessed
using Shapiro–Wilk’s test. For comparison, ANOVA was ap-
plied to the data which was followed by Bonferroni Post-Hoc
test using Graph Pad Prism (Version 9.0) software. All the
data were expressed as the mean ± SEM.
RESULTS
Percent wound contraction, and histological analysis of the
wound scar was performed time by time during the experi-
mental protocol to evaluate the effect of diabetes and various
extracts in the healing of wounds in control and treatment
groups. The healing of the diabetic wound was also evaluated
by the quantification of various biomarkers in the blood of
mice in the control and treatment groups.
Wound Contraction Assessment
The healing area of mice in the control and treatment groups
on different days is represented as percent wound contraction
in Figure 2.
In the last column, the pictures of fully healed wounds
of all the groups are inserted along with the days on which
they healed completely. The non-diabetic control (Normal
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group = N) was healed completely on day 15. On the other
hand, the wound of diabetic control (DC) was fully healed
on day 19. The PC that was treated with ointment (polyfax)
showed complete healing on day 17. Likewise, all the other
groups treated with various extracts and their combinations
showed complete healing sooner than the control groups.
Among all, the best results were shown by group (Ga+Tu+Fi)
in which the combination of garlic (150 mg/ml), turmeric
(100 mg/ml) and fibroin (50 mg/ml) was used and showed
almost complete healing on day 12 as mentioned in Figure 2.
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Percent Wound Contraction on Day 3
A slight significant difference was seen in percent contraction
between the non-diabetic (normal) and DC on the 3rd day (P
< .001 ANOVA). But a significant difference was observed in
percent wound contraction on the 3rd day between the com-
bination of garlic, turmeric, and fibroin (Ga+Tu+Fi) and DC
(P < .001 ANOVA) as Figure 3.
Percent Wound Contraction on Day 7
A significant difference was seen between the DC and all
extracts, their combination groups as a whole at day 7. All the
other groups differed significantly from the DC as evident in
the graph (P < .001 ANOVA) in Figure 3.
Percent Wound Contraction on Day 11
A significant difference was observed between DC and extract,
their combination groups on day 11. The maximum percent
contraction (97.3 ± 2.2%) was observed in group (Ga+Tu+Fi)
treated with the combination of garlic (150 mg/ml), turmeric
(100 mg/ml) and fibroin (50 mg/ml), to DC and PC (P <
.001 ANOVA) as Figure 3.
Histological Analysis
The histological examination of all groups on the 10th day was
illustrated in Figure 4. In the histological slides, neutrophils,
keratinocytes, fibroblast cells, collagen fibers, and blood vessels
were observed at 10×. The maximum number of neutrophils
were observed in the DC group, ultimately involved in delayed
wound healing. But in the normal group (N), collagen fibers
were not properly deposited and fibroblasts were also less
in number. The epithelial layer of the PC group (polyfax)
was also not properly formed on the 10th day. The group
Ga+Tu+Fi showed the best results among all these groups.
The epithelial layer of this group showed properly arranged a
large number of collagen fibers, fibroblasts, keratinocytes, and
blood vessels as compared to all other groups.
Biochemical Parameters
Pro-inflammatory Cytokines. In current studies, three types of
pro-inflammatory cytokines that are, TNF-α, IL-6, and IL-8
were evaluated from blood serum after the complete wound
healing of every group. Although, their levels are maximum
in the inflammatory phase and start decreasing at the end of
healing.
Tumor Necrosis Factor-α (TNF-α). When the values of
TNF-α for all nine groups were compared with the DC
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Figure 2. Pictures of wound of mice of different treatment groups at 0, 5th, 10th day and the day when respective group healed completely.

Figure 3. Comparison of percent wound contraction between the treatment and control groups at 3rd, 7th, and 11th day. ‘a’ indicates the significant differ-
ence between N and DC group, ‘b’ shows the significant difference between DC and PC group, ‘c’ represents the significant difference between DC and Ga
treatment group, ‘d’ designates the significant difference between DC and Tu treatment group, ‘e’ represents the significant difference between DC and
Fi treatment group, ‘f’ shows the significant difference between DC and Ga+Fi treatment group, ‘g’ represents the significant difference between DC and
Tu+Fi treatment group, ‘h’ indicates the significant difference between DC and Ga+Tu treatment group, ‘i’ represents the significant difference between DC
and Ga+Tu+Fi treatment group. Statistical signs: a = P ≤ .05, aa = P ≤ .01, aaa = P ≤ .001. Abbreviations: N = Normal (Non-diabetic) (Saline), D C=Diabetic
Control (Saline), PC =Positive Control (Polyfax), Ga = Garlic Extract (150 mg/ml), Tu = Turmeric Extract (100 mg/ml), Fi = Fibroin (50 mg/ml), Ga+Fi
= Garlic Extract (150 mg/ml) and Fibroin (50 mg/ml), Tu+Fi = Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml), Ga+Tu = Garlic Extract (150
mg/ml) and Turmeric Extract (100 mg/ml), Ga+Tu+Fi = Garlic Extract (150 mg/ml), Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml).
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Figure 4. H & E staining showing the histological differences in diabetic mice skin at 10th post wounding day in different treatment groups.
Magnification of 10X. Scale bar = 100 µm. Abbreviations: N = Normal (Non-diabetic) (Saline), DC = Diabetic Control (Saline), PC = Positive
Control (Polyfax), Ga = Garlic Extract (150 mg/ml), Tu = Turmeric Extract (100 mg/ml), Fi = Fibroin (50 mg/ml), Ga+Fi = Garlic Extract (150
mg/ml) and Fibroin (50 mg/ml), Tu+Fi = Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml), Ga+Tu = Garlic Extract (150 mg/ml) and
Turmeric Extract (100 mg/ml), Ga+Tu+Fi = Garlic Extract (150 mg/ml), Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml).

group, showed an overall statistically significant difference
from the DC (F9,40 = 21.5; P < .0001). The DC group
showed a maximum level (40.9 ± 4.9 pg/ml) of TNF-α
and the minimum level (14.2 ± 0.7 pg/ml) was shown by
the group Ga+Tu+Fi treated with the combination of garlic
(150 mg/ml), turmeric (100 mg/ml) and fibroin (50 mg/
ml) as Figure 5a.
Interleukin-6 (IL-6). When the values of IL-6 for all
groups were compared, they showed statistically significant
differences (F9,40 = 54.4; P < .0001). The maximum level for
IL-6 (28.0 ± 1.8 pg/ml) was measured in the DC group and
the minimum level (10.0 ± 1.1 pg/ml) was determined by
the group Ga+Tu+Fi treated with the combination of garlic
(150 mg/ml), turmeric (100 mg/ml) and fibroin (50 mg/
ml) as Figure 5b.
Interleukin-8 (IL-8). When the values of IL-8 for all
groups were compared, they showed statistically significant
differences (F9,40 = 65.2; P < .0001). The highest level for
IL-8 (60.2 ± 2.4 pg/ml) was shown by the DC group and
the lowest level (17.0 ± 1.0 pg/ml) by the group Ga+Tu+Fi
treated with the combination of garlic (150 mg/ml), turmeric
(100 mg/ml) and fibroin (50 mg/ml). This group Ga+Tu+Fi
showed good results for diabetic wound healing as well as a
maximum significance difference from the DC group (P <
.0001) as Figure 5c.
Matrix Metalloproteinases (MMPs). In current studies, three
types of matrix metalloproteinases that are, MMP2, MMP7,
and MMP9 were evaluated from blood serum after the com-
plete healing of every group. Although their levels are max-
imum in the proliferation phase the level must be low at the
end of healing for the proper remodeling phase.
Matrix Metalloproteinases 2 (MMP2). Overall, a significant
difference was shown among the values of MMP2 for all ten
groups (F9,40 = 22.4; P < .0001). The minimum value for
MMP2 was determined in group Tu+Fi (228.2 ± 10.6 pg/
ml) and group Ga+Tu+Fi (228.0 ± 18.1 pg/ml), these groups
were treated with the combination of two extracts turmeric
(100 mg/ml) and fibroin (50 mg/ml), and the combination
of garlic (150 mg/ml), turmeric (100 mg/ml) and fibroin
(50 mg/ml), respectively. These values showed maximum
difference with the DC group. The best results were shown
by group Ga+Tu+Fi as compared with other parameters like
MMP2 and MMP9 in Figure 6a.
Matrix Metalloproteinases 7 (MMP7). Overall, a significant
difference was shown among the values of MMP7 for all
ten groups (F9,40 = 16.5; P < .0001). The minimum value
(271.4 ± 9.9 pg/ml) for MMP7 was determined in a group
(Ga+Tu+Fi) treated with the combination of garlic (150 mg/
ml), turmeric (100 mg/ml) and fibroin (50 mg/ml), and the
maximum level (454.6 ± 20.5pg/ml) were determined by the
DC group. This group Ga+Tu+Fi showed a maximum differ-
ence from the DC group. The best results were shown by this
group in Figure 6b.
Matrix Metalloproteinases 9 (MMP9). A significant difference
was shown among the values of MMP9 for all ten groups
(F9,40 = 32.3; P < .0001). The minimum value (141.8 ± 5.3
pg/ml) for MMP9 was observed in the group treated with
the combination of garlic (150 mg/ml), turmeric (100 mg/
ml) and fibroin (50 mg/ml), and the maximum level
(370.0 ± 13.5 pg/ml) was determined by the DC group.
This group Ga+Tu+Fi showed a maximum difference from
the DC group. The best results were shown by this group in
Figure 6c.
Tissue Inhibitors Matrix Metalloproteinases (TIMPs). The level
of TIMPs must be increased during the remodeling phase
of the wound healing for the inactivation of MMPs. But in
the diabetic mice, the TIMPs level was measured minimum
(51.8 ± 7.7 pg/ml). The group Ga+Tu+Fi was treated with
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Figure 5. Serum level of pro-inflammatory cytokines among different groups after the complete healing in mice (A) TNF-α, (B) IL-6, and
(C) IL-8. ‘a’ indicates the significant difference between N and DC group, ‘b’ shows the significant difference between DC and PC group, ‘c’
represents the significant difference between DC and Ga treatment group, ‘d’ designates the significant difference between DC and Tu treatment
group, ‘e’ represents the significant difference between DC and Fi treatment group, ‘f’ shows the significant difference between DC and Ga+Fi
treatment group, ‘g’ represents the significant difference between DC and Tu+Fi treatment group, ‘h’ indicates the significant difference between
DC and Ga+Tu treatment group, ‘i’ represents the significant difference between DC and Ga+Tu+Fi treatment group. Statistical signs: a = P ≤
.05, aa = P ≤ .01, aaa = P ≤ .001. Abbreviations: N = Normal (Non-diabetic) (Saline), DC = Diabetic Control (Saline), PC = Positive Control
(Polyfax), Ga = Garlic Extract (150 mg/ml), Tu = Turmeric Extract (100 mg/ml), Fi = Fibroin (50 mg/ml), Ga+Fi = Garlic Extract (150 mg/ml)
and Fibroin (50 mg/ml), Tu+Fi = Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml), Ga+Tu = Garlic Extract (150 mg/ml) and Turmeric
Extract (100 mg/ml), Ga+Tu+Fi = Garlic Extract (150 mg/ml), Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml).
 Journal of Burn Care & Research
8  Farooq et al XXXX/XXXX 2022

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Figure 6. Serum level of MMPs and TIMPs among different groups after the complete healing in mice (A) MMP2, (B) MMP7, (C) MMP9, and
(D) TIMPs. ‘a’ indicates the significant difference between N and DC group, ‘b’ shows the significant difference between DC and PC group, ‘c’
represents the significant difference between DC and Ga treatment group, ‘d’ designates the significant difference between DC and Tu treatment
group, ‘e’ represents the significant difference between DC and Fi treatment group, ‘f’ shows the significant difference between DC and Ga+Fi
treatment group, ‘g’ represents the significant difference between DC and Tu+Fi treatment group, ‘h’ indicates the significant difference between
DC and Ga+Tu treatment group, ‘i’ represents the significant difference between DC and Ga+Tu+Fi treatment group. Statistical signs: a = P ≤
.05, aa = P ≤ .01, aaa = P ≤ .001. Abbreviations. N = Normal (Non-diabetic) (Saline), DC = Diabetic Control (Saline), PC = Positive Control
(Polyfax), Ga = Garlic Extract (150 mg/ml), Tu = Turmeric Extract (100 mg/ml), Fi = Fibroin (50 mg/ml), Ga+Fi = Garlic Extract (150 mg/ml)
and Fibroin (50 mg/ml), Tu+Fi = Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml), Ga+Tu = Garlic Extract (150 mg/ml) and Turmeric
Extract (100 mg/ml), Ga+Tu+Fi = Garlic Extract (150 mg/ml), Turmeric Extract (100 mg/ml) and Fibroin (50 mg/ml).

a combination of garlic (150 mg/ml), turmeric (100 mg/
ml) and fibroin (50 mg/ml), and showed the highest
value (193.0 ± 9.5 pg/ml). Overall, a significant difference
was shown among the values of TIMPs for all ten groups
(F9,40 = 20.7; P < .0001) as Figure 6d.
DISCUSSION
As reported, garlic, turmeric, and fibroin possess wound
healing properties. The antibacterial activity of garlic was
observed against various gram-negative and gram-positive
bacterium.41–44 For healing diabetic wounds, aged garlic
solutions (AGS) were used in chicken wound healing. AGS ac-
celerated the healing process by re-epithelialization and angio-
genesis.12 Allicin, a component of garlic, enhances the wound
process by activating fibroblast cells.13
Turmeric has the potential for wound healing due to its
anti-inflammatory,16 anti-infectious,15 and antioxidant14 ac-
tivities. Curcumin also promotes cutaneous wound healing
because it involves tissue remodeling, collagen deposition,
tissue formation, and granulation.18,45 Other components of
turmeric like turmerones, elemene, furanodiene, Curdione,
bisacurone, and germacrone, also have anti-inflammatory,20,21
antioxidant,22,23 and antimicrobial24–26 activities. That’s
why, turmeric was used for the treatment of diabetic wound
healing.27 The mice wounds treated with curcumin showed
20% more contraction compared to the control.46
In the wound healing process, silk fibroin promotes cell
growth, migration, and proliferation.28 Fibroin also promotes
the various other processes involved in wound healing like cel-
lular adhesion, re-epithelialization, wound contraction, col-
lagen formation, and angiogenesis.47–49 Silk fibroin suppresses
the pro-inflammatory cytokines and also deactivates the apop-
totic pathways of the cells in the inflammatory phase.28
The extract of biomaterial that is, garlic, turmeric, and fi-
broin, accelerated wound healing in diabetic mice. These
extracts showed a good result as compared to DC (saline) and
PC (polyfax). But the combination of these extracts showed
the best results compared to these extracts. The last and fourth
combination of garlic (150 mg/ml), turmeric (100 mg/ml)
and fibroin (50 mg/ml) extract increased the healing process
to the maximum level as compared to all other combinations
of the two extracts. It healed the diabetic wound in 12 days
and also showed the best results as compared to DC (saline)
Journal of Burn Care & Research
Volume XX, Number XX
and PC (polyfax). All extracts possess healing capabilities, in
combination, these extracts collectively enhanced the healing
process and reduced the period of wound healing.
As discussed earlier, these three extracts have individually
good wound healing activities. Various extracts when applied
in combinations to the wound showed best results than indi-
vidual extracts.50 Various researchers used the combinations
of different plant extracts which promoted the healing in
their combinations concerning the individual extracts. The
combination of sodium alginate, extracted from Sargassum
duplicatum, and okra fruit extract promoted diabetic wound
healing in mice.51 Two other biomaterials, Centella asiatica
and Carica papaya extracts were used individually as well as
in the combination with various concentrations for healing
in mice. The combinations accelerated the healing process
more effectively.52 For wound healing, Eclipta alba, Centella
asiatica, and Piper nigrum extracts were used with the com-
bination of two extracts and the combination of these three
extracts in albino rats. The combination of these three
extracts showed maximum healing as compared to the other
combinations.53 In the current studies, the combination of
garlic extract, turmeric extract, and fibroin increased diabetic
wound healing in mice. Among all combinations, the best
results were observed by the combination of garlic (150 mg/
ml), turmeric (100 mg/ml) and fibroin (50 mg/ml).
As reported, these three biomaterials’ garlic, turmeric,
and fibroin increase the number of fibroblast cells and col-
lagen fiber at the wound healing site along with the angio-
genesis.12,13,28,45,47,49,54 In the present studies, the increase in
fibroblasts, and collagen fibers along with the angiogenesis
were observed in the histological slides of all extracts and their
combinations as compared to the DC and PC (polyfax).
The level of various biomarkers is also disturbed in di-
abetes which affects wound healing.55 The level of pro-
inflammatory cytokines that are, TNF-α, IL-6, and IL-8, is
increased in diabetic conditions and involved in inflamma-
tion.56 Matrix metalloproteinases (MMPs) that are, MMP2,
MMP7, and MMP9, and their inhibitor tissue inhibitor ma-
trix metalloproteinases (TIMPs) level is disturbed in diabetes.
Their disturbed ratio causes impaired wound healing.57,58 The
level of pro-inflammatory cytokines that is, TNF-α, IL-6, and
IL-8, was controlled by using various biomaterials.59–62 In the
same way, the level of MMPs was decreased and TIMPs were
increased by using biomaterials.63–65 In the present studies, the
biomaterials, and their combination lowered the level of the
pro-inflammatory cytokine that are, TNF-α, IL-6, and IL-8,
along with the MMPs that are, MMP2, MMP7, and MMP9
increased the TIMPs. This regulation contributed to the ac-
celeration of wound healing in the other treatment groups to
DC. The best results for the regulation of biomarkers were
observed in the group which was treated with the combina-
tion of garlic (150 mg/ml), turmeric (100 mg/ml) and fi-
broin (50 mg/ml).
CONCLUSION
In diabetic mice, the process of wound healing can be accel-
erated by using various biomaterials. In current studies, three
biomaterials that is, garlic, turmeric, and fibroin were used to
Farooq et al  9
heal diabetic mice. These biomaterials are applied individu-
ally and also their combination. The combination of 150 mg/
ml garlic (Allium Sativum), 100 mg/ml turmeric (Curcuma
longa), and 50 mg/ml fibroin showed the maximum %
wound contraction on the 11th day as compared to DC.
This combination also showed a large number of fibroblasts,
keratinocytes, collagen fibers, and blood vessels in histological
slides. The level of various biomarkers, which are involved in
wound healing, was also regulated in the best way by the com-
bination of these three biomaterials. In this way, the diabetic
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wound can be cured by using various biomaterials.
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