# Influence of Honey on Energy Metabolism during  Wound Healing in Rats

Authors: M. Sumitra; P. Manikandan; V. S. Gayathri; L. Suguna
Journal: Scholarly Research Exchange
 (2009)

---

## Abstract

The aim of this study is to find out how honey influences the activities of the enzymes involved in energy metabolism during wound healing. Carbohydrate metabolism is the main source of energy for skin cells during wound healing. Since honey is full of sugars, and hence a rich source of energy, we have investigated the efficacy of honey on energy metabolism during wound healing in rats. A total number of 48 animals were used. From these, 24 animals were divided into two groups, control and experimental. Full thickness excision wounds were made on the back of rats. The control rats were left untreated. The experimental rats received 500μL of honey topically, once daily, for 12 days. Six animals from each group were sacrificed on different time point intervals like 4, 8, and 12 days. The remaining 12 animals were used to evaluate the contraction and epithelialization. The activities of hexokinase, phosphofructokinase glyceraldehyde-3-phosphate dehydrogense, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase were measured in the granulation tissue. A marked increase in the activities of all the glycolytic enzymes in the experimental wounds when compared to control suggests that honey could provide sufficient energy for cellular activity needed for the repair process.


---

## Body

## 1. Introduction

Wound healing represents a dynamic physiological process initiated and
influenced by many factors. This complex cascade of event starts from the moment
of injury and can continue for varying periods of time depending on the extent of
wounding. The process can be broadly categorized into three stages; inflammatory
phase (consisting of the establishment of homeostasis and inflammation); proliferate
phase (consisting of granulation, contraction, and epithelialization); finally
the remodeling phase which ultimately determines the strength and
appearance of the healed tissue [1].Honey is widely
accepted as an excellent adjuvant for wound healing [2].
Honey has been used in the treatment of burns and wounds for many centuries,
with documents describing this use dating back to 1700 BC [3, 4]. Honey acts mainly as a hyperosmolar medium
and prevents bacterial growth [5–7]. Because of its high viscosity, it forms a physical
barrier, and the presence of enzyme catalase gives honey an antioxidant
property [8, 9]. Its
high nutrient content improves substrate supply in local environment promoting
epithelialization and angiogenesis [10, 11].The skin cells
undergo rapid cell division during the process of wound repair and this is
accompanied by a greater demand of metabolic energy. The skin presents an
active site for carbohydrate metabolism [12]. It
has been demonstrated that the proliferating epithelial cells as well as
wounded skin are primarily dependent on the glycolytic pathway for energy requirement
[13–15]. Since honey has been used for wound healing, since time immemorial,
and is a rich source of energy, we have investigated the role of honey on the
glycolytic enzyme activities during wound healing in rats.


## 2. Materials and Methods

### 2.1. Animals

Male
Wistar rats weighing 150–200 g were used
for this study. The animals were maintained on clean, sterile, polyvinyl cages
and fed with commercial rat feed from M/s Hindustan Unilever Limited, Mumbai, India
(mixed with wheat flour in the ratio of 1:1 (w/w)). Food and water were
providedad libitum to the animals. All procedures were carried out
according to the stipulations of the Institutional Animal Care and Use
Committee (IACUC).


### 2.2. Chemicals

Natural honey was
procured from the Dabur India Limited, Uttar Pradesh, India. Remaining chemicals were
purchased from Sigma Chemical Co., St
Louis, Mo, USA.


### 2.3. Wound Creation, Animal Grouping, and Drug Administration

Rats weighing 150–200 g were used for the study. The fur on the
back of the rats was shaved under light ether anaesthesia. Subsequently, full
thickness open excision type of wounds of standard size (2 cm2,
square shaped) was made by removing the skin using a sterile blade [16]. A
total of 36 animals were divided into two groups (control and experimental groups). The control rats
were left untreated, whereas the experimental rats received 500 μL of unprocessed natural honey topically, once daily, for a period
of 12 days. Six rats from each group were sacrificed at different time point
intervals. The wound tissues from the center of the wounds were removed on the 4th, 8th,
and 12th days postwounding and used for the enzyme assays.To find out the rate of contraction and period of
epithelialization of wounds, a separate group of animals (six animals per
group) were maintained. 500μL of honey was
administered topically to the experimental group once daily and the control
group was left untreated. The rate of contraction was measured planimetrically
by tracing the wound area on a transparent graph paper. Period of
epithelialization was measured as number of days taken for the complete healing
of wounds so that no raw wound is left behind.


### 2.4. Preparation of Tissue Homogenate

Samples for the estimation of enzyme activities associated with
energy metabolism were prepared by the method described by Gupta et al. [17]. All operations were performed at 4°C unless
mentioned specifically. Briefly, a 20% homogenate was made in chilled 0.15 M KCl
containing 5 mM EDTA using Polytron Homogenizer. The granulation tissues were
homogenized by giving four strokes of 15 seconds duration for 2 minutes. Samples were
centrifuged at 9000×g in a refrigerated centrifuge for 20 minutes to collect
the supernatant.The assays of all the enzymes were performed at room temperature.
The methodology was designed to ensure maximal enzyme activities in the
homogenate.


### 2.5. Assay of Enzyme Activity

#### 2.5.1. Hexokinase

Hexokinase (HK, E.C.2.7.1.1) activity was measured by the method
of Supowit and Harris [18], using glucose as a substrate. The sample was mixed
with assay buffer consisting of 50 mM triethanolamine and 10 mM MgCl2 (pH
7.4), 1 M glucose, 50 mM NADP, and 140 U/mL of glucose-6-phosphate dehydrogenase.
The reaction was initiated by the addition of 200 mM ATP, and the rate of NADP
reduction was recorded at 340 nm for 3 minutes. Calculation of enzyme activity
was based on the reduction of NADP.


#### 2.5.2. Phosphofructokinase

Phosphofructokinase (PFK, E.C.2.7.1.11) activity was measured by
the method of Bergmeyer et al. [19], using fructose-6-phosphate as a
substrate. The sample was added to 50 mM Tris-HCl buffer (pH 8.0), 500 mM MgCl2, 1 M KCl, 100 mM ATP, 100 mM fructose-6-phosphate, 10 mM NADH, 50 mM
phosphoenolpyruate, and 75 U/mL of pyruvate kinase with 40 U/mL of lactate
dehydrogenase. The NADH metabolized was assessed at 340 nm. PFK activity was
calculated based on 1 μmol fructose-6-phosphate = 2 μmol NADH consumed.


#### 2.5.3. Glyceraldehyde-3-Phosphate Dehydrogenase

The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C.1.2.1.12)
was measured by the method of Kirk and Ritz [20]. The enzyme sample
was mixed with 15 mM pyrophosphate buffer (pH 8.4), 30 m M NAD, and 0.4 M sodium
aresenate. The reaction was initiated by the addition of 0.42 M glyceraldehydes
solution. The NAD metabolized was read at 340 nm for 5 minutes. The enzyme
activity was expressed as units/mg protein under incubation conditions. Protein
was estimated by the method of Lowry et al. [21].


#### 2.5.4. Lactate Dehydrogenase

Lactate dehydrogenase (LDH, E.C.1.1.1.27) activity was estimated
according to the method of King [22]. Lactate was used as the substrate. The
enzyme sample was mixed with substrate and NAD to start the reaction. The
reaction mixture was incubated at 37°C for 15 minutes. 2,4-dinitrophenyl
hydrazine was added to arrest the reaction. The absorbance was measured at 420 nm. The enzyme activity was expressed as nmoles of pyruvate formed/mg
protein/min.


#### 2.5.5. Glucose-6-Phosphate Dehyrogenase

The activity of glucose-6-phosphate dehyrogenase (G6PD, E.C.1.1.1.49) was measured by the method of Lohr and Waller [23].
Glucose-6-phosphate was used as the substrate. The enzyme sample was mixed with
50 mM triethanolamine buffer, 5 mM EDTA, and 30 mM NADP. Reaction was initiated
by the addition of 42.6 mM glucose-6-phosphate. The increase in O.D. at 340 nm was
measured for 2 minutes at every 30 seconds interval.


### 2.6. Statistical Analysis

All results were expressed as mean ± SD. Statistical significance between two
groups was determined by student'st-test
and P<.05 was considered as significant.


## 2.1. Animals

Male
Wistar rats weighing 150–200 g were used
for this study. The animals were maintained on clean, sterile, polyvinyl cages
and fed with commercial rat feed from M/s Hindustan Unilever Limited, Mumbai, India
(mixed with wheat flour in the ratio of 1:1 (w/w)). Food and water were
providedad libitum to the animals. All procedures were carried out
according to the stipulations of the Institutional Animal Care and Use
Committee (IACUC).


## 2.2. Chemicals

Natural honey was
procured from the Dabur India Limited, Uttar Pradesh, India. Remaining chemicals were
purchased from Sigma Chemical Co., St
Louis, Mo, USA.


## 2.3. Wound Creation, Animal Grouping, and Drug Administration

Rats weighing 150–200 g were used for the study. The fur on the
back of the rats was shaved under light ether anaesthesia. Subsequently, full
thickness open excision type of wounds of standard size (2 cm2,
square shaped) was made by removing the skin using a sterile blade [16]. A
total of 36 animals were divided into two groups (control and experimental groups). The control rats
were left untreated, whereas the experimental rats received 500 μL of unprocessed natural honey topically, once daily, for a period
of 12 days. Six rats from each group were sacrificed at different time point
intervals. The wound tissues from the center of the wounds were removed on the 4th, 8th,
and 12th days postwounding and used for the enzyme assays.To find out the rate of contraction and period of
epithelialization of wounds, a separate group of animals (six animals per
group) were maintained. 500μL of honey was
administered topically to the experimental group once daily and the control
group was left untreated. The rate of contraction was measured planimetrically
by tracing the wound area on a transparent graph paper. Period of
epithelialization was measured as number of days taken for the complete healing
of wounds so that no raw wound is left behind.


## 2.4. Preparation of Tissue Homogenate

Samples for the estimation of enzyme activities associated with
energy metabolism were prepared by the method described by Gupta et al. [17]. All operations were performed at 4°C unless
mentioned specifically. Briefly, a 20% homogenate was made in chilled 0.15 M KCl
containing 5 mM EDTA using Polytron Homogenizer. The granulation tissues were
homogenized by giving four strokes of 15 seconds duration for 2 minutes. Samples were
centrifuged at 9000×g in a refrigerated centrifuge for 20 minutes to collect
the supernatant.The assays of all the enzymes were performed at room temperature.
The methodology was designed to ensure maximal enzyme activities in the
homogenate.


## 2.5. Assay of Enzyme Activity

### 2.5.1. Hexokinase

Hexokinase (HK, E.C.2.7.1.1) activity was measured by the method
of Supowit and Harris [18], using glucose as a substrate. The sample was mixed
with assay buffer consisting of 50 mM triethanolamine and 10 mM MgCl2 (pH
7.4), 1 M glucose, 50 mM NADP, and 140 U/mL of glucose-6-phosphate dehydrogenase.
The reaction was initiated by the addition of 200 mM ATP, and the rate of NADP
reduction was recorded at 340 nm for 3 minutes. Calculation of enzyme activity
was based on the reduction of NADP.


### 2.5.2. Phosphofructokinase

Phosphofructokinase (PFK, E.C.2.7.1.11) activity was measured by
the method of Bergmeyer et al. [19], using fructose-6-phosphate as a
substrate. The sample was added to 50 mM Tris-HCl buffer (pH 8.0), 500 mM MgCl2, 1 M KCl, 100 mM ATP, 100 mM fructose-6-phosphate, 10 mM NADH, 50 mM
phosphoenolpyruate, and 75 U/mL of pyruvate kinase with 40 U/mL of lactate
dehydrogenase. The NADH metabolized was assessed at 340 nm. PFK activity was
calculated based on 1 μmol fructose-6-phosphate = 2 μmol NADH consumed.


### 2.5.3. Glyceraldehyde-3-Phosphate Dehydrogenase

The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C.1.2.1.12)
was measured by the method of Kirk and Ritz [20]. The enzyme sample
was mixed with 15 mM pyrophosphate buffer (pH 8.4), 30 m M NAD, and 0.4 M sodium
aresenate. The reaction was initiated by the addition of 0.42 M glyceraldehydes
solution. The NAD metabolized was read at 340 nm for 5 minutes. The enzyme
activity was expressed as units/mg protein under incubation conditions. Protein
was estimated by the method of Lowry et al. [21].


### 2.5.4. Lactate Dehydrogenase

Lactate dehydrogenase (LDH, E.C.1.1.1.27) activity was estimated
according to the method of King [22]. Lactate was used as the substrate. The
enzyme sample was mixed with substrate and NAD to start the reaction. The
reaction mixture was incubated at 37°C for 15 minutes. 2,4-dinitrophenyl
hydrazine was added to arrest the reaction. The absorbance was measured at 420 nm. The enzyme activity was expressed as nmoles of pyruvate formed/mg
protein/min.


### 2.5.5. Glucose-6-Phosphate Dehyrogenase

The activity of glucose-6-phosphate dehyrogenase (G6PD, E.C.1.1.1.49) was measured by the method of Lohr and Waller [23].
Glucose-6-phosphate was used as the substrate. The enzyme sample was mixed with
50 mM triethanolamine buffer, 5 mM EDTA, and 30 mM NADP. Reaction was initiated
by the addition of 42.6 mM glucose-6-phosphate. The increase in O.D. at 340 nm was
measured for 2 minutes at every 30 seconds interval.


## 2.5.1. Hexokinase

Hexokinase (HK, E.C.2.7.1.1) activity was measured by the method
of Supowit and Harris [18], using glucose as a substrate. The sample was mixed
with assay buffer consisting of 50 mM triethanolamine and 10 mM MgCl2 (pH
7.4), 1 M glucose, 50 mM NADP, and 140 U/mL of glucose-6-phosphate dehydrogenase.
The reaction was initiated by the addition of 200 mM ATP, and the rate of NADP
reduction was recorded at 340 nm for 3 minutes. Calculation of enzyme activity
was based on the reduction of NADP.


## 2.5.2. Phosphofructokinase

Phosphofructokinase (PFK, E.C.2.7.1.11) activity was measured by
the method of Bergmeyer et al. [19], using fructose-6-phosphate as a
substrate. The sample was added to 50 mM Tris-HCl buffer (pH 8.0), 500 mM MgCl2, 1 M KCl, 100 mM ATP, 100 mM fructose-6-phosphate, 10 mM NADH, 50 mM
phosphoenolpyruate, and 75 U/mL of pyruvate kinase with 40 U/mL of lactate
dehydrogenase. The NADH metabolized was assessed at 340 nm. PFK activity was
calculated based on 1 μmol fructose-6-phosphate = 2 μmol NADH consumed.


## 2.5.3. Glyceraldehyde-3-Phosphate Dehydrogenase

The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C.1.2.1.12)
was measured by the method of Kirk and Ritz [20]. The enzyme sample
was mixed with 15 mM pyrophosphate buffer (pH 8.4), 30 m M NAD, and 0.4 M sodium
aresenate. The reaction was initiated by the addition of 0.42 M glyceraldehydes
solution. The NAD metabolized was read at 340 nm for 5 minutes. The enzyme
activity was expressed as units/mg protein under incubation conditions. Protein
was estimated by the method of Lowry et al. [21].


## 2.5.4. Lactate Dehydrogenase

Lactate dehydrogenase (LDH, E.C.1.1.1.27) activity was estimated
according to the method of King [22]. Lactate was used as the substrate. The
enzyme sample was mixed with substrate and NAD to start the reaction. The
reaction mixture was incubated at 37°C for 15 minutes. 2,4-dinitrophenyl
hydrazine was added to arrest the reaction. The absorbance was measured at 420 nm. The enzyme activity was expressed as nmoles of pyruvate formed/mg
protein/min.


## 2.5.5. Glucose-6-Phosphate Dehyrogenase

The activity of glucose-6-phosphate dehyrogenase (G6PD, E.C.1.1.1.49) was measured by the method of Lohr and Waller [23].
Glucose-6-phosphate was used as the substrate. The enzyme sample was mixed with
50 mM triethanolamine buffer, 5 mM EDTA, and 30 mM NADP. Reaction was initiated
by the addition of 42.6 mM glucose-6-phosphate. The increase in O.D. at 340 nm was
measured for 2 minutes at every 30 seconds interval.


## 2.6. Statistical Analysis

All results were expressed as mean ± SD. Statistical significance between two
groups was determined by student'st-test
and P<.05 was considered as significant.


## 3. Results

Table1
depicts the HK activity at different time point intervals. There is a marked
increase (more than 100%) in the enzyme activity in honey-treated wounds, particularly on the 8th day wherein the
fibroblast proliferation and collagen synthesis are actively taking place.Table 1
Hexokinase activity of control and honey-treated wounds.
Day postwoundingControlExperimental4890 ± 16.11200 ± 19.0***81450 ± 24.23010 ± 29.5***12920 ± 10.11400 ± 13.6***Enzyme activity is expressed as nmoles/min/g of wet tissue.Values are expressed as mean ± SD from six rats in each individual experiment.P*** <.001 as compared to control.The differences in the PFK activity are depicted in Table2. There is more than twofold increase in this
enzyme activity in honey-treated wounds.Table 2
Phosphofructokinase activity of control and honey-treated wounds.
Day postwoundingControlExperimental411.20±0.9325.21±2.65***813.20±1.0130.21±3.12***1214.12±1.9330.32±3.35***Enzyme activity is expressed as nmoles/min/g of wet tissue.Values are expressed as mean ± SD from six rats in each individual experiment.P*** <.001 as compared to control.Table3
shows the GAPDH activity in the control and experimental wounds. There is about
75% increase in this enzyme activity in honey-treated wounds when compared to
the control.Table 3
Glyeraldehyde-3-phosphate dehydrogenase activity of control and honey-treated wounds.
Day postwoundingControlExperimental46.41±1.1711.12±1.42***811.51±0.9220.12±1.02***129.41±1.9115.12±1.52***Enzyme activity is expressed as units/mg protein/g of wet tissue.Values are expressed as mean ± SD from six rats in each individual experiment.P*** <.001 as compared to control.Tables4 and 5 depict the LDH and G6PDH activities on the control and experimental
wounds, respectively. There is a 114% and 68% increase in LDH and G6PDH,
respectively, in honey-treated wounds as compared to control. The enzyme levels
start increasing on the 4th day, reach the maximum on the 8th day and decline
on the 12th day.Table 4
Lactate dehydrogenase activity of control and honey-treated wounds.
Day postwoundingControlExperimental4120 ± 23300 ± 38***8200 ± 12429 ± 29***12160 ± 12380 ± 42***Enzyme activity is expressed as
nmoles of pyruvate formed/min/mg protein.Values are expressed as mean ± SD from six rats in each individual experiment.P*** <.001 as compared to control.Table 5
Glucose-6-phosphate dehydrogenase activity of control and honey-treated wounds.
Day postwoundingControlExperimental467.2±6.5101.4±7.9***889.2±4.5150.4±9.9***1286.2±6.5141.9±3.2***Enzyme activity is expressed as nmoles/min/g of wet tissue.Values are expressed as mean ± SD from six rats in each individual experiment.P*** <.001 as compared to control.Figure1 shows the photographical representation of the contraction rate of wounds at
different time point intervals. From the pictures, we could observe that the
contraction is much faster in honey-treated wounds (data also given as Table 6). Figure 2 shows the period of epithelialization wherein we could observe
that the experimental wounds took 12±1 day for complete epithelialization
when compared to control wounds which took 22±2 days.Table 6
Rate of contraction as percent of original wound size of control and honey-treated
wounds at different days.
GroupsExperimental days4812Control16 ± 240 ± 375 ± 5Honey treated38 ± 3***72 ± 5***99 ± 1***Values are expressed in mean ± SD from six animals.P***<.001 as compared to control.Rate of contraction as percentage of original wound size of
control and experimental wounds—scale 1 cm.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 2
Values are expressed in mean ± SD for six animals.*P<.01 as compared to control.


## 4. Discussion

In normal skin, glucose utilization provides energy equally through glycolysis and
the mitochondrial system. But in wounded regenerating skin, especially in the
migrating epithelium, glucose utilization provides energy mainly through
glycolytic pathway [12]. Hence, measuring the glycolytic enzyme activities may
be a useful indicator of the degree of wound repair.Honey is a natural product which contains almost 80% sugars. The energy needed for
the collagen synthesis may be provided by these sugars, which enter the
glycolytic pathway. The glycolytic pathway is the main source of energy for
fibroblasts, which synthesize collagen [12, 24]. Lactate, the end product of
glycolysis, increases prolyl hydroxylase activity so as to prepare the
fibroblasts for an increased rate of hydroxylation of proline and collagen extrusion
after the fibroblasts are exposed to oxygen [25].Before the new capillary network forms, there is a marked gradient of oxygen within
the wound [26] with the center of the wound being the most deficient in oxygen
[27, 28]. Therefore, the increased activities of the glycolytic enzymes
indicate that the connective tissue cells, probably fibroblasts, in the early
stages of wound healing have metabolically adopted for the survival in a
hypoxic environment. The increase in G6PDH and glycolytic enzymes may indicate
that glucose is utilized by the repairing tissue to provide both energy in the
form of ATP and certain basic substances for the increased cellular activities
of biosynthesis, mitosis, and locomotion [12].The significant increase in lactate content, the end product of glycolysis,
increases the prolyl hydroxylase activity so as to prepare the fibroblasts for
an increased rate of hydroxylation of proline and collagen extrusion after the
fibroblasts are exposed to oxygen [25]. The increased rate of contraction and
epithelialization in honey-treated wounds may be due to its hygroscopic property
which enables it to dehydrate bacteria, rendering them inactive to dehydrate edematous
wounds, reducing their surface area and making them more clearly defined.
Dehydration of unwanted fluids around the wound might also decrease tissue
turgor and improve tissue oxygenation and hence wound healing [29].The acidic pH of honey [30] and its hypertonicity also helps the wounds to heal
fast. In a double blind study, Kaufman et al. [31] applied buffered
solutions to experimental second degree wound burns in guinea pigs and noted
significantly increased reepithelialization in wounds treated with a solution of pH
3.5 compared with wounds treated with neutral and alkaline solutions. Bergman et al. [32] showed that honey increases the
thickness of the granulation tissue and the area of reepithelialization, when applied
topically.Gupta et al. [17, 33] have reported the
importance of energy metabolism during wound healing in diabetic and
immunocompromised and aged conditions. The results of the present investigation
suggest that honey could enhance wound healing by accelerating the glycolytic
enzyme activities, making
sufficient energy available for cellular activity needed for the repair
process.



---

*Source: 715320-2009-03-03.xml*