Natural extracts and honey based impregnated gauze wound dressing preparation and in vitro antibacterial efficacy

 

Wissam Zam1*, Rim Harfouch2, Rand Ali1, Yara Atfah1, Amena Mousa2

1Department of Analytical and Food Chemistry, Faculty of Pharmacy, Al-Andalus University for Medical Sciences, Tartous, Syrian Arab Republic.

2Department of Microbiology, Faculty of Pharmacy, Al-Andalus University for Medical Sciences, Tartous, Syrian Arab Republic.

*Corresponding Author E-mail: w.zam@au.edu.sy

 

ABSTRACT:

A wide range of wound dressings is commonly introduced to target different aspects of the wound healing process. The ideal dressing should maintain a rapid healing at low cost with minimal inconvenience to the patient. Pharmacological agents such as antibiotics, anti-inflammatory and other wound healing accelerators are added to dressings in order to improve the properties of wound dressings. Honey, pomegranate peels and olive leaves extracts are suitable natural products with a wide range of antibacterial and healing accelerator properties. Impregnated gauzes containing starch based mixtures of honey and aqueous extracts of olive leaves or pomegranate peels were prepared and their antibacterial efficacy was studied. Different prepared mixtures possess an antibacterial activity against both Pseudomonas aeruginosa and Staphylococcus aureus comparable to synthetic tested antibiotics. Impregnated gauze prepared using starch based mixtures of honey and pomegranate peel extract were more potent against S. aureus compared with impregnated gauze prepared using starch based mixtures of honey and olive leaves extract. The prepared impregnated gauze does not need a secondary dressing and can be cut to fit around the wound due to their flexible nature. Soft elastic property of impregnated gauze provides easy application and removal after wound is healed without any damage.

 

KEYWORDS: Impregnated gauze, Honey, Pomegranate peel aqueous extract, Olive leaves aqueous extract, Pseudomonas aeruginosa, Staphylococcus aureus.

 

 


 

INTRODUCTION:

A wound is defined as a disruption in the continuity of the epithelial lining of the skin or mucosa resulting from physical or thermal damage. According to the duration and nature of healing process, the wound is categorized as acute and chronic1,2. The key to successful wound healing is careful wound care and the optimization of the bodys wound-healing capacity. Numerous dressing products and topical medications were developed continuously to accelerate the healing process. Cho and Lo reported that dressings increase re-epithelialization rates and collagen synthesis -by about 30% to 50% compared to wounds exposed to air3. Additionally all wounds are contaminated with bacteria at the time they occur but there are sufficient body defenses to protect the patient from invasion by these contaminating bacteria. However, there are a number of wounds which become so heavily infected at the time they are obtained that the large number of bacteria present overcomes the resistance of the individual and serious infections ensue. It was reported that the application of topical antibiotic decreases bacterial counts, whereas systemic antibiotics have no role in the management of wounds unless there is evidence of cellulitis or sepsis4. Bacteria have the ability to adapt to environmental conditions and resist to antibiotics, after overcoming and neutralizing immune barriers, followed by multiplication and invasion of the host organism. The frequency of drug-resistant bacteria is increasing and includes original drug-sensitive bacteria. According to Prvnescu et al. in a study performed on 470 samples of wound secretions with positive bacteriological diagnosis, Staphylococcus aureus and Pseudomonas aeruginosa were both greatly resistant to many antibiotics5.

 

Natural products provide a wide field of research and an approved alternative for antibacterial treatment. Honey is carbohydrate-rich syrup used in wound care due to its healing properties associated with its antibacterial and anti-inflammatory activities6. Antibacterial activity of honey is due to its pH, osmolarity, hydrogen peroxide production, and phytochemicals originated from the nectar of plant7. Honey maintains a moist wound condition, and its high viscosity helps to provide a protective barrier to prevent infection8. Its acidity increases the release of oxygen from hemoglobin thereby making the wound environment less favorable for the activity of destructive proteases and the high osmolarity of honey draws fluid out of the wound bed to create an outflow of lymph as occurs with negative pressure wound therapy9. Hydrogen peroxide is produced by dilution with body fluids and acts as an antiseptic10. The application of honey can promote the healing in infected wounds that do not respond to the conventional antibacterial therapy, including wounds infected with MRSA or multi-drug resistant Pseudomonas aeruginosa11-14. In Australia and New Zealand, honey is applied as a wound dressing under controlled and sterilized conditions15. In addition, a blend of Manuka and jelly bush honey is medically certified and licensed as medical product for wound care in Europe, America and Australia16,17.

 

The pomegranate (Punicagranatum L.) fruit has been used for centuries in ancient cultures for its medicinal purposes.Pomegranate peel is recognized for its wound healing properties18, antimicrobial activity19-22 and antioxidative capacities23. The peel extract showed highest activity against bacteria and fungi compared to other extracts. Amongst the selected bacterial and fungal cultures, the highest anti-microbial activity was recorded against Staphylococcus aureus24. The efficacy is mainly attributed to the water-soluble polyphenols, anthocyanins and hydrolysable tanninswhich can degrade the cell wall, disrupt the cytoplasmic membrane, damage membrane proteins and interfere with membrane-integrated enzymes, which may eventually lead to cell death25. In addition, the aqueous peel extract has shown potent dermal effect on cultures of human skin cells, stimulating dermal fibroblast proliferation and collagen synthesis while inhibiting major collagen degrading enzyme26.

 

Olive trees (Oleaeuropaea L.) are widely grown in many regions of Syria, while Syrians use olive oil and olive leaf extract for treatment of skin diseases and wounds. The main component of olive leaf extract is Oleuropein which is rich in polyphenols, an anti-inflammatory agent27.Oleuropein, known as an antioxidant, reduces cellular damage to a minimum allowing improvement of wound healing. Mehraein et al. had proved that Oleuropein exerted positive effect on type1 collagen fiber synthesis which confer strength to tissue and increase the rate of epithelialization28.Wound healing efficacy as well as the antioxidant activity of the aqueous extract in vivo wound models was greater compared to the n-hexane extract29. It was also reported that phenolic compounds in olive leaves possess in vitro activity against several Gram negative and Gram positive namely Bacillus cereus, B. subtilis,Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia; and fungi such as Candida albicans and Cryptococcus neoformans30.

 

The function of gauze pads is to prevent from contamination with outside dirt and debris but the common gauze pad offers no protection against the development and multiplication of bacteria in and around the wound surfaces. The aim of our study is to prepare Punicagranatum peel extract and honey impregnated gauze and to study the in vitro antimicrobial activity of the prepared impregnated gauze was studied against the most types of microbial infection occurring in chronic wounds and resistant to a wide range of antibiotics,  Pseudomonas aeruginosa  and  Staphylococcus aureus

 

MATERIALS AND METHODS:

Impregnated gauze preparation

Pomegranate peel aqueous extract was prepared by pouring 1 g of the dried and ground peels in 100 ml of DI water. The mixture was placed in a thermostatic water bath shaker at 50C for 20 min then centrifuged at 2000 rpm for 10 min31.

Olive leaves used in this study were collected from Syrian coastal region. Leaves were washed to remove impurities such as dust, dried in an air for 7 days and then they were ground by grinder. One hundred milliliter of ethanol 40% was added to 2 grams powder obtained from leaves and extracted at 60 C for 120 min32. Finally, ethanol was evaporated using a rotary evaporator and the aqueous extract was used to prepare the impregnated gauze.

 

A starch based gel was first formed using a mixture of starch, glycerol, and water. Each solution was mixed by gentle stirring with a magnetic stir bar until starch dissolved. It was then homogenized and heated for about 15 min at 80-85C.The solution was cooled to room temperature then pomegranate peel or olive leaves aqueous extract and Nigella sativahoney was added with different mixing ratios as presented in table 1.

 


 

Table 1: Starch based gel containing pomegranate peel or olive leaves aqueous extract and honey at different mixing ratios.

Mixture number

Starch (g)

Glycerin (g)

Pomegranate peel aqueous extract (ml)

Olive leaves aqueous extract (ml)

Honey (ml)

1

2

2

ــــــــــ

ــــــــــ

10

2

2

4

ــــــــــ

10

ــــــــــ

3

2

2

ــــــــــ

5

5

4

2

2

ــــــــــ

2.5

7.5

5

2

4

ــــــــــ

7.5

2.5

6

2

4

10

ــــــــــ

ــــــــــ

7

2

2

5

ــــــــــ

5

8

2

2

2.5

ــــــــــ

7.5

9

2

4

7.5

ــــــــــ

2.5

 

 

 


The standard sterile 3 inch by 3 inch was dipped into the working solution till saturation and the excess solution was extruded by applying pressure. The hardening of the gel on the gauze was accomplished by refrigeration then the prepared impregnated gauzes were placed in sterile envelopes. 

 

Antibacterial efficacy

Antimicrobial activity test was carried out using agar diffusion method on Muller Hinton Agar plates33. Plates were inoculated by spreading a volume of Pseudomonas aeruginosa (Table 2) or Staphylococcus aureus (Table 3) isolated from infected wounds routinely submitted to the department of medical microbiology at AL-Andalus University Hospital. One hundred microliter of each mixture was introduced into a hole punched into plates with a diameter of 6 mm. The plates were incubated for 24 h at 37 C and the zone diameter of inhibition was measured at two cross sectional points and the average was taken as the inhibition zone.

 


 

 

 

 

Table 2: Antibiotic sensitivities of P. aeruginosa isolate

Result

Inhibition zone diameter

Antibiotic name

Antibiotic symbol

Sensitive

32 mm

Cefoperazone/Sulbactam

CPS

Sensitive

29 mm

Levofloxacin

LEV

Sensitive

29 mm

Ceftazidime

CAZ

Sensitive

28 mm

Cefadroxil

CFR

Sensitive

25 mm

Cefoperazone

CPZ

Sensitive

24 mm

Imipenem

IMP

Sensitive

20 mm

Chloramphenicol

CHL

Sensitive

20 mm

Amikacin

AMK

Intermediate

21 mm

Cefotaxime

CTX

Intermediate

19 mm

Ceftriaxone

CTR

Intermediate

13 mm

Doxycycline

DOX

Resistant

No inhibition zone

Gentamycin

CN

Resistant

No inhibition zone

Cefuroxime

CXM

Resistant

No inhibition zone

Amoxicillin+ clavulanic acid

AUG

Resistant

No inhibition zone

Spectinomycin

SPC

 

 

 

 

 

 

Table 3: Antibiotic sensitivities of S. aureus isolate

Result

Inhibition zone diameter

Antibiotic name

Antibiotic symbol

Sensitive

31 mm

Imipenem

IMP

Sensitive

30 mm

Levofloxacin

LEV

Sensitive

23 mm

Erythromycin

ERY

Sensitive

22 mm

Meropenem

MER

Sensitive

20 mm

Tetracycline

TET

Sensitive

19 mm

Chloramphenicol

CHL

Resistant

13 mm

Cefotaxime

CTX

Resistant

12 mm

Cefuroxime

CXM

Resistant

11 mm

Linezolid

LNZ

Resistant

10 mm

Cefazolin

CZ

Resistant

9 mm

Ceftazidime

CAZ

Resistant

9 mm

Cefpodoxime

CPD

Resistant

No inhibition zone

Ceftriaxone

CRO

Resistant

No inhibition zone

Cefdinir

CDR

Resistant

No inhibition zone

Cefaclor

CCL

 

 

 

 


RESULTS AND DISCUSSION:

Infection is one of the most frequent complications of wound healing and accounts for considerable patient morbidity, discomfort and prolonged hospitalization. Infection must be avoided or treated with great care to permit proper healing34.

 

As presented in Table 4, starch based mixture containing N. sativa honey at 10% (mixture N. 1) was more effective in inhibiting P. aeruginosa than S. aureus with diameter zones of inhibition 18 cm and 11 cm, respectively. It has been reported from various clinical studies that honey act as a barrier preventing wounds from becoming infected, preventing cross-infection, and allowing burn wound tissue to heal rapidly uninhibited by secondary infection35. Lindberg et al. found that honey was more efficacious for wound healing than silver, and turned out to have more antibacterial qualities36. Some studies also suggest that the topical use of honey may reduce odours, swelling and scarring when used to treat wounds; it may also prevent the dressing from sticking to the healing wound37.

 

Results presented in Table 4 revealed that different mixtures containingolive leaves aqueous extract was effective against both P. aeruginosa and S. aureus. Sudjana et al. had previously proved that commercial olive leaf extract have an antibacterial effect against  Staphylococcusaureus, as well as against methicillin-resistant S. aureus (MRSA)38. In another report olive leaf extracts succeeded in eliminating the Gram-negative P. aeruginosa39. In the present work, it was also noticed that the starch based mixture N. 2 containing only olive leaves extract was more effective against P. aeruginosa than S. aureus. These results were in accordance with Youssef et al. who found that P. aeruginosa and Klebsiella spp. were more sensitive to olive leaves extract as Gram-negative bacteria and E. faecalis among Gram-positive bacteria and that S. aureus strains were the least sensitive among all tested bacteria40.Bouderba et al. also investigated the antibacterial activity of wild olive leaves aqueous extract in Algeria. They found that the diameter zone of inhibition was 25.01 mm for P. aeruginosa and 9.88 mm for S. aureus which is in accordance with our results for mixture N. 241.

 

Combinations between olive leaves extract and honey at different ratios (Mixtures N. 3, 4 and 5) have good efficacy against both P. aeruginosa than S. aureus with a diameter zone of inhibition ranging from 14-15 mm and 11-14 mm, respectively.

 

As noticed in Table 4, starch based mixture N. 6 containing only pomegranate peel extract has similar activity against both P. aeruginosa andS. aureus with a diameter zone of inhibition 20 mm and 19 mm respectively. Our results were in accordance with Bharaniet al.42and Gaber et al.43who found that the diameter zone of inhibition of aqueous pomegranate peel extract ranged from 20-30 mm against bothP. aeruginosa andS. aureus. Same results were obtained by Al-Wazni et al against S. aureus bacteria isolated from urine samples collected from pregnant women suffering from recurrent urinary tract infections44. Combinations between pomegranate peel extract and honey at different ratios (Mixtures N. 7, 8 and 9) have good efficacy against both P. aeruginosa than S. aureus with a diameter zone of inhibition ranging from 14-18 mm.

 

In general and as noticed in Table 4 mixtures containing pomegranate peel extract (mixtures 6, 7, 8 and 9) were more potent than mixtures prepared using olive leaves extract (mixtures 2, 3, 4 and 5) against both P. aeruginosa and S. aureus.

 

 

 

Table 4: Sensitivity of P. aeruginosa and S. aureus isolates against different mixtures

Mixture number

Diameter zone of inhibition (mm)

P. aeruginosa

S. aureus

1

18

11

2

17

12

3

15

11

4

14

11

5

14

14

6

20

19

7

17

18

8

14

15

9

18

18

 

 

Gauze dressings are made from cotton, rayon polyester or a combination of both. Sterile gauze pads are used for packing open wounds to absorb fluid and draw it away from the wound45. Though gauze dressings can provide some bacterial protection, this is lost when the outer surface of the dressing becomes moistened either by wound exudate or external fluids45. In addition gauze dressings tend to become more adherent to wounds as fluid production diminishes and are painful to remove, thus causing patient discomfort46.A randomized trial comparing paraffin gauze and a hydrocolloid dressing applied to skin draft donor sites showed that the hydrocolloid achieves faster healing and is a less painful dressing47. When applied to the wound as a gel, hydrogel dressings usually require a secondary covering such as gauze and need to be changed frequently48.

 


 

Figure 1: Impregnated gauze.

 

 


Impregnated gauze with 10 ml honey.b. Impregnated gauze with 5 ml pomegranate peel aqueous extract and 5 ml honey mixture. c. Impregnated gauze with 5 ml olive leaves aqueous extract and 5 ml honey mixture.

 

As presented in Figure 1, the prepared impregnated gauze however, does not need a secondary dressing and can be cut to fit around the wound due to their flexible nature. Soft elastic property of impregnated gauze provides easy application and removal after wound is healed without any damage.It is used to deliver active agents to wounds and the incorporated natural extracts play an active role in the wound healing process either due to their anti-inflammatory or antimicrobial properties.The high water content of starch based mixtures helps to maintain the granulation tissues and epithelium in a moist environment49. Temperature of cutaneous wounds is decreased by starch mixture providing soothing and cooling effect. The prepared impregnated gauze could be used for dry infected wounds, but are not suitable for heavy drainage wounds.

 

CONCLUSION:

Prevention and treatment of infection in burn wounds are major aims of treatment. Many systemic and topical regimes are in use, but none is completely satisfactory. The prepared impregnated gauzes provide a moist environment that helps wounds epithelialize more rapidly. In addition, honey and aqueous extracts of olive leaves or pomegranate peels provide antibacterial and anti-inflammatory properties that will accelerate the healing process of wounds.

 

It is well known that no one antibiotic is capable of antagonistic action against all Gram-positive and Gram-negative bacteria. Further research on the efficacy of a proper combination of an antibiotic and the impregnated gauze containing honey and the aqueou0s extract of pomegranate peel or olive leaves would be valuable and could be a good alternative for the antibacterial resistant bacterial strains.

 

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Received on 13.10.2017 Modified on 19.11.2017

Accepted on 21.12.2017 A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2018; 10(1): 01-07.

DOI: 10.5958/0975-4385.2018.00001.8